WO2024022911A1 - Antisense oligonucleotides for treating a disease or condition associated with an abnormal processing of app - Google Patents

Antisense oligonucleotides for treating a disease or condition associated with an abnormal processing of app Download PDF

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WO2024022911A1
WO2024022911A1 PCT/EP2023/070010 EP2023070010W WO2024022911A1 WO 2024022911 A1 WO2024022911 A1 WO 2024022911A1 EP 2023070010 W EP2023070010 W EP 2023070010W WO 2024022911 A1 WO2024022911 A1 WO 2024022911A1
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oligonucleotide
seq
nucleotides
wings
base
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Sonia NAJAS SALES
Dennnis Christian FRANKEN
Inmaculada SANJUAN RUIZ
Bart De Strooper
Lutgarde Serneels
Judith Van Deutekom
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Vico Therapeutics B.V.
Katholieke Universiteit Leuven
Vib Vzw
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N2320/34—Allele or polymorphism specific uses

Definitions

  • the invention relates to the field of human genetics, more specifically to treatments for a disease or condition associated with an abnormal (or altered) processing of the Amyloid Precursor Protein (APP), preferably familiar Alzheimer disease (FAD).
  • APP Amyloid Precursor Protein
  • FAD Alzheimer disease
  • the invention in particular relates to antisense oligonucleotides (AONs) that can be used for treating such diseases or conditions.
  • AONs antisense oligonucleotides
  • Neurocognitive disorders include a broad category of degenerative brain diseases marked by a progressive decline in memory, learning, perception, social, recognition, orientation, language, comprehension, and judgment (American Psychiatric Association, 2013, 5 th . Ed. Arlington). Neurocognitive disorders are a manifestation of impaired multiple molecular pathways and cellular functions, leading to synapse loss, cell death, inflammation, gliosis, and disruption of the functional networks that underlie cognition, sensation, and motor skills (Elahi & Miller, 2017 Nature Reviews. Neurology, 13:457-476). The continuous decline in physical function in patients ultimately leads to loss of autonomy and death.
  • AD Alzheimer's disease
  • AD is the most common neurocognitive and neurodegenerative disease, and the most frequent cause of dementia (60% to 80% of cases).
  • AD involves both monogenic and complex forms of inheritance, and is characterized by the accumulation of insoluble amyloid-p plaques (also called Ap plaques) in extracellular spaces and vascular walls, the aggregation of the microtubule protein Tau in neurofibrillary tangles in neurons, dystrophic neurites, loss of synapses, neurotransmitter alterations (acetylcholine deficiency and glutamate excitotoxicity), and micro- and astrogliosis. Molecular and cellular pathological events start decades before dementia becomes apparent.
  • AD Alzheimer's disease
  • Familial Alzheimer’s disease is an Early-Onset AD (EOAD) (before 65 years of age) caused by mutations in genes involved in A peptide production: APP (OMIM 104760), PSEN1 (OMIM 104311), or PSEN2 (OMIM 1600759) (Lanoiselee et al., 2017, PLoS Medicine, 14:3).
  • FAD has an autosomal dominant inheritance pattern and a prevalence of 1 % of all AD cases.
  • Ap plaques depositions are detected in asymptomatic patients and most likely drive the progression of Tau pathology. High rates of Tau accumulation can be seen in symptomatic FAD patients and Tau pathology has been suggested as an indicator of the onset of cognitive impairments (Gordon et al., Brain. 2019 Apr 1 ;142(4):1063-1076).
  • Ap plaques deposition does not correlate with the degree of cognitive decline but may be required for progression of Tau pathology.
  • Ap is produced by sequential cleavage of p-amyloid precursor protein (APP) by p-secretase and y-secretase.
  • the p-secretase enzyme (BACE1) cleaves APP at the N terminus of the Ap sequence, releasing secreted APP-p and the membrane-bound C99 fragment.
  • BACE1 p-secretase enzyme
  • the y-secretase complex binds to N-terminally cleaved APP fragment (C99) and intramembranous cleaves at the s-site releasing C-terminal fragment (CTF) and Ap49 or Ap48.
  • the y-secretase complex then processes along the remaining Ap C-terminal end, producing sequentially shorter peptides until the Ap peptide is released from the complex (generally after producing peptides 38-, 40-, and 42-amino acids in length). Therefore, the processivity of the y-secretase complex defines the length of the generated Ap peptide.
  • Ap peptides are prone to aggregate into p sheet conformations in the form of higher-order oligomers, protofibrils, and fibrils, which are detectable in AD brain. Longer Ap peptides (> Ap42) are more hydrophobic and show more capacity to aggregate and form Ap plaques (L. Chavez-Gutierrez and M.
  • the y-secretase complex consists of four protein subunits: Presenilin (PSEN), presenilin enhancer 2 (PEN-2), Anterior pharynx-defective (APH), and Nicastrin.
  • Presenilin PSEN
  • PEN-2 presenilin enhancer 2
  • APH Anterior pharynx-defective
  • Nicastrin There are multiple isoforms of PSEN (PSEN1 or PSEN2) and APH (APH1A or APH1 B); therefore up to four different y- secretase complexes may exist in a single cell (De Strooper., 2003 Neuron, 38, 9-12).
  • PSEN1 is an aspartyl protease and is the catalytic subunit of y-secretase, PEN-2 is required for y-secretase maturation, APH stabilizes the complex and Nicastrin is thought to play a role in substrate binding (De Strooper et al., 1998, Nature 391 , 387-390, De Strooper et al., 1999, Nature 398, 518-522, Wolfe at al., 1999, Nature 398, 513-517; N. Takasugi et al., 2003, Nature 422, 438-441 ; S. Shah et al., 2005, Cell 122, 435-447; R. Zhou et al., 2019, Science 363, 6428).
  • the proteins involved in Ap peptide production form part of a membrane-embedded protease complex called the y-secretase complex and are composed of the following four proteins with a 1 :1 :1 :1 stoichiometry: Nicastrin (NCT), Anterior pharynx-defective 1 (APH1A or APH1 B), Presenilin enhancer 2 (PEN-2) and Presenilin (PSEN-1 or PSEN-2).
  • Presenilin-1 and -2 are the catalytic subunits of the complex (De Strooper., 2003Neuron, 38, 9-12).
  • Presenilin 1 and Presenilin 2 are paralogs, y-secretase complexes exhibit broad substrate specificity with close to 100 type 1 membrane proteins listed as potential substrates (Wakabayashi et al., 2008, Physiology 23, 194-204).
  • One of the substrates of the y-secretase complex is the Amyloid Precursor Protein (APP), which is converted into Ap peptides by said complex (De Strooper et al., 1998, Nature 391 , 387-390).
  • APP Amyloid Precursor Protein
  • APP may be processed through two distinct pathways: the non- amyloidogenic pathway and the amyloidogenic pathway.
  • AD patients mainly comprising cholinesterase inhibitors and NMDA inhibitors (Long and Holtzman et al., 2019, Cell 170(2), 312- 339).
  • FDA U.S. Food and Drug Administration
  • Adulhelm is a monoclonal antibody that targets extracellular Ap plaques in the brain. (https://www.fda.gov/news-events/press- announcements/fda-grants-accelerated-approval-alzheimers-drug)
  • APP Amyloid Precursor Protein
  • FAD Alzheimer disease
  • the inventors designed antisense oligonucleotides that could be used for such treatment. Such antisense oligonucleotides are expected not to have all drawbacks of existing treatments.
  • A PSEN1 protein levels
  • B-D gamma-secretase activity
  • AON20 SEQ ID NO:1 18
  • a -B Western blot analysis showing the protein levels of full-length PSEN1 (A) or the protein levels of CTF APP normalized by full-length APP (B) after 8 days treatment with AON20 at the indicated concentrations. Data in A and B is shown relative to the non-treated condition (OpM).
  • C-D ELISA analysis showing the profile of soluble Ap38, Ap40 and Ap42 species in the supernatant of mouse Tg/Tg A431 E neurons after 8 days treatment with AON20 at the indicated concentrations.
  • dd PCR analysis showing mRNA levels of WT and A431 E PSEN1 in human neurons differentiated from patient-derived iPSC after 10 days of treatment with AON14 (SEQ ID NO:28), AON15 (SEQ ID NO:30), and AON16 (SEQ ID NO:33).
  • A mouse Psenl mRNA levels
  • B-D y-secretase activity
  • AON87 targets specifically mouse WT Psenl .
  • A RT-qPCR analysis showing the mRNA levels of A431 E and WT mouse Psenl after 7 days of treatment with AON87 at the indicated concentrations. mRNA levels of A431 E and WT Psenl were normalized to the levels of two housekeeping genes (Actin and Rsp23). Data is shown relative to the non-treated condition (OpM).
  • the invention targets the real underlying source of FAD by depleting the mutant form of the allele and in that way restoring the normal activity of the catalytic subunit of the y-secretase complex using an oligonucleotide of the invention as defined herein.
  • Such oligonucleotide preferentially targets and breaks down the allele carrying a mutation in PSEN1 or PSEN2, which will reduce the levels of mutant PSEN1 or PSEN2, allow replacement by the corresponding wild type proteins, increase the levels of functional y -secretase, and thus reduce the production of toxic forms of Ap peptides.
  • This strategy is different from attempts to inhibit or downregulate the y-secretase as such which causes severe (Notch-)signaling side effects and is also expected to be more effective than current approaches that focus on more downstream features of the disease such as Tau aggregation or more generic features of the disease like inflammation for the treatment of familial AD.
  • This strategy is quite unique: the oligonucleotides are selected to have the highest possible efficiency and the highest possible specificity against the mutated allele of PSEN1 or PSEN2, while they are selected for the lowest possible efficiency and the lowest possible specificity against the wild type allele of PSEN1 or PSEN2. It means the oligonucleotides have been designed to specifically inactivate, delete, knock down, suppress the mutated allele of the PSEN1 or PSEN2 and primarily keep the wild type allele thereof intact.
  • APP Amyloid Precursor Protein
  • antisense oligonucleotides are described below in more detail. Such antisense oligonucleotides will be referred to herein as antisense oligonucleotides according to the invention (or antisense oligonucleotide of the invention or oligonucleotide of the invention or oligonucleotide or AON).
  • an antisense oligonucleotide which preferentially targets a mutated allele of a protein of the y-secretase complex when present in a cell comprising (preferably expressing) said mutated allele.
  • the protein of the y- secretase complex whose allele is mutated is PSEN1 or PSEN2, preferably PSEN1 .
  • the antisense oligonucleotide is single-stranded.
  • an antisense oligonucleotide comprising 15 to 30 or 20 to 30 nucleotides including a central region of 5 to 15 consecutive DNA nucleotides and/or DNA nucleotides analogues flanked in each end by wing regions comprising 1 to 5 RNA nucleotides analogues, which preferentially targets a mutated allele of a protein of the y-secretase complex when present in a cell comprising and preferably expressing said mutated allele, wherein the mutation present in the mutated allele is targeted by the first, second, third, fourth, fifth, sixth, seventh, eight, nineth, tenth, eleventh, or twelfth nucleotide of the central region of the oligonucleotide.
  • the protein of the y-secretase complex whose allele is mutated is PSEN1 or PSEN2, preferably PSEN1.
  • the antisense oligonucleotide is single-stranded.
  • the antisense oligonucleotides of the second aspect are demonstrated to efficiently target a mutated allele of a protein of the y- secretase complex, such as PSEN1 .
  • an antisense oligonucleotide comprising 15 to 30 or 20 to 30 nucleotides including a central region of 5 to 15 consecutive DNA nucleotides and/or DNA nucleotides analogues flanked in each end by wing regions comprising 1 to 5 RNA nucleotides analogues, which preferentially targets a mutated allele of a protein of the y-secretase complex when present in a cell comprising and preferably expressing said mutated allele, wherein the mutation present in the mutated allele is targeted by the sixth, seventh, eighth, nineth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, or eighteenth nucleotide in the oligonucleotide sequence starting from 5’ to 3’.
  • the protein of the y-secretase complex whose allele is mutated is PSEN1 or PSEN2, preferably PSEN1.
  • the antisense oligonucleotide is single-stranded.
  • said cell is a cell, wherein said protein is endogenously expressed (mutated allele and preferably the wild type allele).
  • the cell is a mammalian cell, preferably a murine or human cell.
  • the cell is representative for the disease FAD.
  • a cell which is representative for the disease FAD is a neuronal cell.
  • Preferred neuronal cells are cells of the central nervous system.
  • a preferred cell of the central nervous system may be from, or may be derived from, a region of the brain which is known to be affected in FAD.
  • brain regions include the hippocampus, amygdala, cerebral cortex (e.g frontal lobe, parietal lobe, temporal lobe and/or occipital lobe) cerebellum, and the thalamus.
  • the activity of the antisense oligonucleotide is assessed in such neuronal cells. The assessment may also be carried out in vitro by culturing such cells and contacting them with the oligonucleotide.
  • a cell which is representative for the disease FAD is a human iPSC (induced pluripotent stem cell), which is triggered to differentiate or mature into neurons.
  • the antisense oligonucleotides of the invention are able to successfully target proteins of the y-secretase complex whose allele is mutated (PSEN1) in biologically relevant FAD systems, such as iPSC cells.
  • the iPSC is from a subject having an altered or diminished PSEN1 or PSEN2 activity, and is a FAD patient or is suspected to become a FAD patient due to his/her genetic background.
  • the activity of the antisense oligonucleotide is assessed using such neuronal cells derived from human iPSC.
  • the assessment may be carried out in vitro by culturing such cells and contacting them with the oligonucleotide.
  • said cell is a cell, wherein said protein is exogenously expressed (mutated allele or the wild type allele).
  • the cell is a mammalian cell, preferably a murine or human cell.
  • a nucleic acid coding for said mutated protein is introduced into said cell.
  • Said cell may be a non-neuronal cell, including but not limited to fibroblast, HeLa, or HEK293 cell.
  • Said nucleic acid coding for said mutated protein may be introduced by viral or non-viral vectors, more preferably plasmids expressing the mutant protein or fusion-derivatives thereof.
  • said cell is a cell wherein said protein is endogenously expressed.
  • a nucleic acid coding for said mutated protein is introduced into said cell.
  • the skilled person may also assess the activity of the oligonucleotide in a cell exogenously and subsequently in a cell endogenously expressing the mutated protein.
  • the choice of the read out is a key feature of the invention as the use of a given read out may lead to the identification of oligonucleotide that are not optimal when later validated using a more relevant read out.
  • the inventors demonstrated that the use of the effect of the oligonucleotide on the mutated PSEN1 or PSEN2 transcript and/or the absence of effect on the wild type allele of said transcript and/or the effect of the oligonucleotide to normalize, reverse or correct abnormal A042/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios, preferably the Ap42/38 and/or Ap43/40 ratios were crucial, more preferably Ap42/38 ratios were crucial.
  • antisense oligonucleotides of the invention have the biggest impact on Ap38 peptide levels and the Ap42/Ap38 ratio while the effect on other Ap-species (such as Ap40) is less pronounced (Examples 2, 6, and 8). This observation confirms the need of using holistic readouts to evaluate the therapeutic benefit of the therapeutic effect of antisense oligonucleotides;
  • an antisense oligonucleotide which preferentially targets a mutated allele of a protein of the y- secretase complex (preferably PSEN1) when present in a cell comprising (preferably expressing) said mutated allele.
  • the cell is a neuronal cell, even more preferably a human neuronal cell.
  • the cell expresses the endogenous mutated allele of a protein of the y-secretase pathway.
  • the oligonucleotide is able to preferentially silence, inactivate, break down, knock down, reduce or decrease mutated PSEN1 or PSEN2 transcripts (preferably mutated PSEN1 transcripts) and even more preferably
  • the oligonucleotide is not (or minimally) able to silence, inactivate, break down, knock down, reduce or decrease wild-type PSEN1 or PSEN2 transcripts (preferably wild-type PSEN1 transcripts).
  • the oligonucleotide is able to preferentially silence, inactivate, break down, knock down, reduce or decrease mutated PSEN1 or PSEN2 transcripts (preferably mutated PSEN1 transcripts), and even more preferably the oligonucleotide is not (or minimally) able to silence, inactivate, break down, knock down, reduce or decrease wild-type PSEN1 or PSEN2 transcripts (preferably mutated PSEN1 transcripts) and/or
  • the oligonucleotide is able to normalize, reverse or correct abnormal Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios, preferably the Ap42/38 and/or Ap43/40 ratios, and more preferably the Ap42/38 ratio.
  • the oligonucleotide :
  • the oligonucleotide :
  • an activity elicited or exhibited by an antisense oligonucleotide of the invention is to preferentially target a mutated allele of a protein of the y-secretase complex when present in a cell comprising said mutant allele.
  • said protein is a human protein and/or is PSEN1 .
  • the targeted mutated allele especially the region or stretch of this mutated allele which is targeted may also be named the targeted region of the oligonucleotide.
  • This mutated allele of a human protein of the y- secretase complex may be a disease-associated or disease-causing allele in a cell of a patient, in a tissue of a patient and/or in a patient as explained later herein.
  • the patient is a mammal.
  • the patient is a human.
  • oligonucleotide is synonymous with the expression “antisense oligonucleotide” in the context of the application.
  • preferentially binds could be used interchangeably when used in the context of an antisense oligonucleotide of the invention which is reverse complementary to a part of a mutated allele, especially in the form of a transcript encoding this mutated allele as identified herein.
  • This “preferential binding/targeting/hybridizing” is later herein defined by comparison to the “specific binding/targeting/hybridizing” to a wild type/control/non mutated allele.
  • mutated allele refers to the pre-mRNA or mRNA encoded by an allele.
  • a mutated allele is a gene encoding a mutated transcript, said mutated transcript encoding a mutated protein of the y- secretase complex.
  • the wild type genomic DNA of mouse PSEN1 comprises SEQ ID NO: 145
  • the corresponding coding RNA comprises SEQ ID NO:146
  • the mouse PSEN1 protein comprises SEQ ID NO:147.
  • the A431 E genomic DNA of mouse A431 E PSEN1 comprises SEQ ID NO: 148
  • the corresponding coding RNA comprises SEQ ID NO:149
  • the mouse A431 E PSEN1 protein comprises SEQ ID NO:150.
  • the wild type genomic DNA of human PSEN1 comprises SEQ ID NO: 151
  • the corresponding coding RNA comprises SEQ ID NO:152 or 169
  • the human PSEN1 protein comprises SEQ ID NO:153 or 159.
  • the A431 E genomic DNA of human A431 E PSEN1 comprises SEQ ID NO: 154
  • the corresponding coding RNA comprises SEQ ID NO:155 or 170
  • the human A431 E PSEN1 protein comprises SEQ ID NO:156 or 161.
  • hybridization refers to the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid/target region). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases).
  • the natural base adenine is nucleobase complementary to the natural nucleobases thymine, 5-methyluracil and uracil which pair through the formation of hydrogen bonds.
  • the natural base guanine is nucleobase complementary to the natural bases cytosine and 5-methyl-cytosine.
  • Hybridization can occur under varying circumstances.
  • hybridization of an oligonucleotide of the invention with a targeted pre-mRNA and/or mRNA can occur under varying circumstances.
  • binding of an oligonucleotide of the invention to a targeted pre-mRNA and/or mRNA i.e. targeted region
  • said hybridization or said binding is assessed under physiological conditions in a cell, more preferably in a human cell.
  • Preferred cells have been defined earlier herein.
  • An oligonucleotide of the invention is preferably said to be able to bind to, or capable of binding to, or able to hybridize with, or capable of hybridizing with, when said binding or hybridization occurs under physiological conditions in a cell, preferably a human cell.
  • Preferred cells have been defined earlier herein.
  • hybridizes or “binds” (or “preferentially hybridizes” or preferentially binds” or “preferentially binds” or “preferentially hybridizes”) is used under physiological conditions in a cell, preferably a human cell unless otherwise indicated.
  • an antisense oligonucleotide is said to target a mutated allele encoding a mutated protein of the y-secretase complex (preferably PSEN1) when said oligonucleotide is reverse complementary to at least 10 or at least 15 consecutive/contiguous bases of said mutated allele or of said mutated transcript encoding said mutated protein.
  • This “at least 10 or at least 15 consecutive/contiguous bases of said mutated allele” may also be called the targeted region or the target region of the antisense oligonucleotide.
  • the length of the reverse complementary part may be at least 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 nucleotides.
  • an oligonucleotide comprises up to 50 nucleotides and/or nucleotide analogues.
  • An oligonucleotide may be 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides and/or nucleotide analogues.
  • the part of the oligonucleotide which is reverse complementary does not per se need to be 100% reverse complementary with the mutated allele or transcript of a mutated protein of the y-secretase complex.
  • one ortwo mismatches may be present over a length of at least 15 nucleotides in total defining the part which is reverse complementary with said mutated allele or transcript.
  • An activity of the oligonucleotide of the invention is to preferentially target a mutated allele or mutated transcript encoding a mutated protein of the y-secretase complex.
  • This activity may mean that (the levels of) the targeted mutant allele (or corresponding transcript) is silenced or knocked down (or broken down) or inactivated or reduced or decreased. In other words, the levels of said mutated transcript are knocked down (or broken down), reduced or decreased.
  • the protein of the y-secretase complex is PSEN1.
  • the protein is a human protein. More preferably, human PSEN1.
  • a first way of assessing an activity of the antisense oligonucleotide of the invention is to assess whether the oligonucleotide is able to knock down (or break down) or reduce or decrease (the levels of) said mutated transcript (preferably PSEN1 or PSEN2 mutated allele or transcript).
  • This reduction or decrease of the level of mutated transcript may be of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the level of the same transcript at the onset of the treatment.
  • the mutated transcript is no longer detectable.
  • the reduction or decrease may be assessed in cells or tissues or subjects treated with said oligonucleotide and using Northern Blotting or (semi) quantitative RT-qPCR or RT-ddPCR analysis of mutant transcript copy numbers (preferably as carried out in the experimental part).
  • the level of the wild type allele or wild type transcript which is not targeted (or not specifically targeted) by the oligonucleotide is also assessed. Accordingly, the wild type allele or transcript is still detectable and the level of said wild type allele or transcript is not less than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the level of the same allele or transcript at the onset of the treatment. In a preferred embodiment, the level of the wild type allele or transcript is the same as the one detected at the onset of the treatment.
  • the presence of said transcript may be assessed in cells or tissues or subjects treated with said oligonucleotide and using Northern Blotting or (semi) quantitative RT-qPCR or RT-ddPCR analysis of mutant transcript copy numbers (preferably as carried out in the experimental part).
  • the activity of the antisense oligonucleotide of the invention is assessed as follows:
  • This reduction or decrease of the level of mutated transcript may be of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the level of the same transcript at the onset of the treatment.
  • the mutated transcript is no longer detectable.
  • the oligonucleotide is said not (or minimally) be able to knock down or reduce or decrease the wild type PSEN1 or PSEN2 allele or transcript when said wild type allele or transcript is still detectable is not less than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the level of the same allele or transcript at the onset of the treatment.
  • the oligonucleotide is able to knock down or reduce or decrease the levels of a mutated PSEN1 or PSEN2 transcript of at least 20 or at least 30 or at least 40 or at least 50 or at least 80 or at least 90 or at least 95% compared to the level of said mutated allele or transcript at the onset of the treatment and the levels of a wild type PSEN1 or PSEN2 transcript is still at least 50 or at least 80 or at least 90 or at least 95% of the level of said transcript of allele at the onset of the treatment.
  • some mutations in PSEN1 or PSEN2 gene may shift y- secretase complex activity towards the production of abnormal Ap profiles, from short peptides (A 38, A 40) to longer peptides (A 42, A 43 and even longer up to A 48 or A 49, although there are no tools readily available to measure those species while the other ones are readily measurable with ELISA assays), as the result of an impairment of the carboxypeptidase-like activity of y-secretase.
  • the underlying mechanism is the destabilization of the substrate-enzyme complex i.e. the APP substrate interaction with the catalytic presenilin subunit in the y-secretase complex is decreased because of the mutation of the PSEN1 or PSEN2 gene.
  • Ap49 or larger or equal to Ap48 larger or equal to Ap42 or larger or equal to Ap43 forms of the Ap peptide species are toxic because they are more hydrophobic and aggregate more easily and form more easily toxic oligomeric and fibrillic species.
  • the processing of Ap is progressive and there are two production lines, one starting with Ap49>46>43>40>37 and one starting at Ap48>45>42>38 (Takami et al., 2009, J Neurosci 29, 13042-52).
  • Ap42/38 and/or Ap 43/40 and/or Ap43/37 and/or Ap 42/40 ratios are expected to increase in brain, cerebrospinal fluid (CSF) and/or plasma of FAD patients because of the PSEN1 or PSEN2 mutations.
  • the Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios is increased of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to the level in healthy subjects.
  • the Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios may be assessed by techniques known to the skilled person.
  • the Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios may be assessed using cell extract, cell medium, brain extract, CSF or plasma of FAD patients carrying PSEN1 or PSEN2 mutations.
  • the Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios may be assessed using cell extract, cell medium of cells endogenously or exogenously expressing a mutated PSEN1 or PSEN2.
  • FAD patients with no detectable Ap plaques in the brain FAD patients with no detectable Ap plaques in the brain may be called asymptomatic FAD patients.
  • Ap plaques are formed and the ratios decreased in CSF samples . In plasma, these ratios are increased during all the progression of the disease. Therefore if the patient already has detectable Ap plaques in the brain, the assay should be carried out in the plasma of said patients.
  • the ELISA assay only assesses the soluble Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio) and the effect of the aggregation process on the level of the ratios cannot be specified. Said ELISA assay primarily visualizes the first effect of the mutation of the y-secretase complex activity, which is the generation of longer forms of the A peptide species.
  • an asymptomatic FAD subject or patient is selected or identified in view of his/her family history with several FAD patients having a PSEN1 or PSEN2 mutation. It is crucial to diagnose this type of subject when they are still asymptomatic (and therefore as early as possible) in order to start treatment as early as possible.
  • the skilled person may use alternative in vitro assays that are able to assess the presence of aggregated A peptides in brain extracts from a mouse.
  • a second way of assessing an activity of the oligonucleotide of the invention is to determine the Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, and more preferably the Ap42/38 ratio) in cell cultures, tissues, plasma and/or CSF of treated FAD subjects.
  • the FAD patients are asymptomatic, with no detectable Ap plaques in the brain.
  • the oligonucleotide is expected to reduce the generation of long Ap species leading to reduction of soluble Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio).
  • the assessment is carried out in subjects, it is preferably carried out in the plasma and/or CSF of said subjects.
  • the normalization, correction, or reversion of abnormal Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios may mean a decrease of the A042/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio) of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to its level at the onset of the treatment.
  • the Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios may be assessed by techniques known to the skilled person. In an embodiment it is assessed using ELISA, preferably as carried out in the experimental part.
  • the assessment is carried out in FAD patients with detectable Ap plaques in the brain, it is preferably carried out in the plasma and not in the CSF of said patients.
  • the invention is uniquely using a read out which directly demonstrates the therapeutic effect of an oligonucleotide on a disease or condition associated with an abnormal (or altered) processing of the APP.
  • This read out directly demonstrates the downstream consequences of the defects of the y-secretase complex present in a cell, in a tissue, in plasma and/or in CSF of a FAD patient by assessing Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio).
  • the FAD patient is preferably asymptomatic with no detectable Ap plaques in the brain, as earlier defined herein.
  • the effect of the oligonucleotide on this read out is expected to be representative of a therapeutic effect on a patient with a mutated y-secretase complex (preferably PSEN1) that will develop FAD. It is expected that the used read out (i.e.
  • Ap42/38 and/or A 43/40 and/or A 43/37 and/or A 42/40 ratios is representative of the FAD disease and the normalization, correction or reversion of the abnormal increased soluble Ap42/38 and/or A 43/40 and/or Ap43/37 and/or A 42/40 ratios (preferably the Ap42/38 and/or A 42/40 ratios, more preferably the Ap42/38 ratio) is therefore predictive of a therapeutic activity of an oligonucleotide.
  • This read out (any of the soluble Ap42/38 and/or A 43/40 and/or A 43/37 and/or A 42/40 ratios (preferably the Ap42/38 and/or A 42/40 ratios, more preferably the Ap42/38 ratio), also called “a long/short” A ratios) is more representative and more predictive than a read out limited to a single “long” A peptide only (such as soluble Ap42).
  • the goal of the therapy is to restore a normal enzymatic activity of the y- secretase complex or to switch the activity of said complex to a more normal activity.
  • long/short Ap peptide ratios provide a better representation of the enzymatic activity of the y-secretase complex than the use of just a single long form of the Ap.
  • the y- secretase complex preferably PSEN1
  • an antisense oligonucleotide of the invention may be considered to be active when:
  • the oligonucleotide is able to normalize, correct, or reverse abnormal A042/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio), wherein the assessment is compared to the level of the same Ap ratio at the onset of the treatment (or to corresponding level of the same Ap ratios in healthy subjects).
  • the assessment may be carried out in a cell, a tissue, plasma, or CSF or subject. When a subject is treated, said ratios are preferably assessed in plasma of said subjects.
  • a subject may be an asymptomatic FAD subject.
  • the subject may be a symptomatic FAD subject. If the subject is a symptomatic FAD subject, the assessment is preferably carried out using plasma.
  • An alternative way of assessing the effect of the oligonucleotide on the Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios is applying imaging technologies to assess Ap plaque accumulation in the brain of treated subjects. Imaging technology may include PET scans on FAD patients. Imaging technology may also include histology on murine tissues.
  • an antisense oligonucleotide of the invention may be considered to be active when:
  • the oligonucleotide is able to preferentially silence, inactivate, breakdown, knock down or reduce or decrease or correct (the quantity or level of) mutated PSEN1 (or PSEN2) transcripts
  • the oligonucleotide is not (or minimally) able to silence, inactivate, breakdown, knock down or reduce or decrease or correct (the quantity or level of) wild-type PSEN1 (or PSEN2) transcripts, and the oligonucleotide is able to normalize, correct, or reverse abnormal A042/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio), preferably wherein the assessment is compared to the level of the same mutant transcript or the same Ap ratio at the onset of the treatment (or to corresponding level of the same Ap ratio in healthy subjects).
  • the assessment may be carried out in a cell, a tissue, plasma, or CSF or subject.
  • a subject When a subject is treated, said ratios are preferably assessed in plasma of said subjects.
  • a subject may be an asymptomatic FAD subject.
  • the subject may be a symptomatic FAD subject. If the subject is a symptomatic FAD subject, the assessment is preferably carried out using plasma.
  • An alternative way of assessing the effect of the oligonucleotide on the Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio) is applying imaging technologies to assess Ap plaque accumulation in the brain of treated subjects. Imaging technology may include PET scans on FAD patients. Imaging technology may also include histology on murine tissues.
  • an antisense oligonucleotide of the invention is considered to be active when: - the oligonucleotide is able to preferentially silence, inactivate, breakdown, knock down or reduce or decrease or correct (the quantity or level of) mutated PSEN1 (or PSEN2) proteins,
  • the oligonucleotide is not (or minimally) able to silence, inactivate, breakdown, knock down or reduce or decrease or correct (the quantity or level of) wild-type PSEN1 (or PSEN2) proteins, and
  • the oligonucleotide is able to normalize, correct, or reverse the abnormal increased soluble A042/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio), preferably wherein the assessment is compared to the level of the same mutant protein orthe same Ap ratio at the onset of the treatment (or to corresponding level of the same Ap ratio in healthy subjects).
  • the assessment may be carried out in a cell, a tissue, plasma, or CSF or subject.
  • a subject may be an asymptomatic FAD subject.
  • the subject may be a symptomatic FAD subject. If the subject is a symptomatic FAD subject, the assessment is preferably carried out using plasma.
  • the silencing or inactivation or breaking down or reduction or decrease or knocking down of the mutated transcript is expected to induce a similar reduction or decrease of the levels of the mutated protein (preferably PSEN1).
  • an antisense oligonucleotide of the invention may be considered to be active when:
  • the oligonucleotide is able to preferentially silence, inactivate, breakdown, knock down or reduce or decrease or correct (the quantity or level of) mutated PSEN1 (or PSEN2) transcripts,
  • the oligonucleotide is not (or minimally) able to silence, inactivate, breakdown, knock down or reduce or decrease or correct (the quantity or level of) wild-type PSEN1 (or PSEN2) transcripts,
  • the oligonucleotide is able to normalize, correct, or reverse abnormal Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the A042/38 and/or Ap42/40 ratios, more preferably the A042/38 ratio) and,
  • the oligonucleotide is able to reduce the formation of Ap plaques, preferably when compared to the level of the same (mutant) transcript or the same Ap ratio or the quantity of Ap plaques at the onset of the treatment (or to corresponding level of the same transcript, or Ap ratio in healthy subjects).
  • the A042/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios may be assessed using ELISA, preferably as carried out in the experimental part.
  • the formation of Ap plaques may be assessed using imaging technologies in the brain of treated subjects. Imaging technology may include PET scans on FAD patients. Imaging technology may also include histology on murine tissues.
  • the assessment may be carried out in a cell, a tissue, plasma, or CSF or subject.
  • a subject When a subject is treated, said ratios are preferably assessed in plasma of said subjects.
  • a subject may be an asymptomatic FAD subject.
  • the subject may be a symptomatic FAD subject. If the subject is a symptomatic FAD subject, the assessment is preferably carried out using plasma.
  • the antisense oligonucleotide of the invention preferentially targets a mutated allele (and not, or minimally, the WT allele) of protein PSEN1 or PSEN2 when present in a cell expressing said mutated allele.
  • Preferred cells have been earlier defined herein.
  • the antisense oligonucleotide of the invention preferentially targets a mutated allele (and not, or minimally , the WT allele) of protein PSEN1 (preferably human protein PSEN1) of the y-secretase complex when present in a cell expressing said mutated allele.
  • PSEN1 preferably human protein PSEN1
  • an antisense oligonucleotide of the invention is said to be active when,
  • the level of the corresponding wild-type allele or transcript is not less than 50%, 60%, 70%, 80%, 90%, 95% or 100% of the level of the same allele or transcript at the onset of the treatment.
  • the allele or transcript is preferably from the PSEN1 or PSEN2 gene, more preferably from the PSEN1 gene.
  • the invention is not limited to a specific mutation found in PSEN1 or PSEN2. There are already several hundred mutations known for PSENI that are linked with FAD.
  • the PSEN1 mutation is selected from the group consisting of the most frequent PSEN1 mutations: P117L, M139T, M139V, M146I, H163R, G206A, P264L, E280A, L392V and A431 E , more preferably E280A and A431 E.
  • Most preferred mutation in PSEN1 is A431 E.
  • the PSEN1 mutation is selected from the group consisting of the most frequent PSEN1 mutations: P117L, M139T, M139V, M146I, H163R, G206A, P264L, E280A, L392V, A431 E, and S212Y, more preferably E280A and A431 E.
  • Most preferred mutation in PSEN1 is A431 E
  • the PSEN1 mutation is selected from the group consisting of the mutations caused by a change of a guanine, thymine or cytosine to an adenosine in the coding sequence resulting in one of the following mutations in the PSEN1 protein: R35Q, V94M, F105I, R108Q, L113Q, T116N, P117T, P117Q, E120K, E123K, M139K, M139I, V142I, M146I, V151 M, Y154N, L166H, L174M, 11 SON, G206S, G206D, G209R, G209E, S212Y, H214N, G217D, S230N, A231T, M233I, F237I, A246E, Y256N, V261 I, G266S, R269H, V272D, T274K, R278K, E280K, R358Q, A360T, S365Y, G
  • the oligonucleotide may be designed as follows: when the base of the nucleotide present in the oligonucleotide and targeting the mutation is a thymine, said thymine is replaced by an uracil.
  • Examples are provided of oligonucleotides targeting the A431 E mutated allele of PSEN1 . Such examples are disclosed in SEQ ID NO: 26, 28, 30, 33, 35, 37, 39.
  • Examples are also provided of oligonucleotides targeting the S212Y mutated allele of PSEN1. Such examples are disclosed in SEQ ID NO: 188, 190, 192, 194, 198, 200.
  • Preferred oligonucleotides targeting this mutation via a thymine have this thymine be replaced by a uracil and this uracil is at the first, second or third position of their central part.
  • Said preferred oligonucleotides targeting this mutation via a thymine have this thymine replaced by a uracil at the sixth, seventh, or eighth nucleotide in the oligonucleotide sequence starting from 5’ to 3’.
  • Other preferred oligonucleotides targeting this mutation via a thymine have this thymine replaced by a uracil and this uracil is at the first, second, third, or seventh position of the central part of the oligonucleotide.
  • Said preferred oligonucleotides targeting this mutation via a thymine have this thymine replaced by a uracil at the sixth, seventh, eighth, or twelfth nucleotide in the oligonucleotide sequence starting from 5’ to 3’.
  • This replacement increases mutant allele selectivity (See examples 3-4), possibly due to a slightly decreased melting temperature (Tm).
  • examples 3 and 4 indicate that antisense oligonucleotides with a thymine-uracil replacement at the above-mentioned preferred nucleotide positions exhibit an increased mutant allele selectivity of PSEN1 .
  • examples are provided of oligonucleotides targeting the A431 E or S212Y mutated allele of PSEN1. Preferred examples are found in SEQ ID NO: 28, 30, 33, 37, 39, 47, 97, 99, 115, 192, 194, 198.
  • central part or “central region” of an antisense oligonucleotide refers to a specific section or segment of the oligonucleotide sequence.
  • the precise definition and boundaries of the central part can vary depending on the context of the invention.
  • the central part may be determined based on the nucleotide position relative to the ends of the oligonucleotide.
  • the central part refers to the distinct region of the gapmer present between two flanking regions/wings.
  • the central part is designed to specifically target a mutated allele of a protein of the y-secretase complex.
  • an oligonucleotide is a polymer of nucleotides and/or a polymer of nucleotides analogues.
  • the expression “nucleotide analogue” may be replaced by “derived from a nucleotide”.
  • An oligonucleotide comprises or consists of repeating monomers. Within the context of the invention, an oligonucleotide comprises up to 50 nucleotides and/or nucleotide analogues.
  • Said oligonucleotide may have 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Such oligonucleotide may also be identified as an oligonucleotide having from 10 to 50 nucleotides or from 12 to 50 nucleotides. Attractive results were obtained with oligonucleotides having a length ranged from 15 to 30 nucleotides, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.
  • the antisense oligonucleotide of the invention comprises nucleotides and/or nucleotides analogues.
  • an antisense oligonucleotide of the invention comprises 15 to 30 nucleotides including a central region of DNA nucleotides (and/or DNA nucleotide analogues) flanked at each end by wing regions comprising RNA nucleotides analogues.
  • the antisense oligonucleotide comprising 15 to 30 nucleotides including a central region of DNA nucleotides flanked at each end by wing regions comprising RNA nucleotides analogues relates to the antisense oligonucleotide of the second aspect of the invention.
  • a DNA nucleotide analogue may be present in the central region of DNA nucleotides as long as the natural recruitment of RNaseH to the AON-target RNA duplex is not significantly reduced or hindered or completely abolished and/or its on target activity is not significantly reduced.
  • an antisense oligonucleotide as defined herein is able to recruit RNaseH to silence, inactivate, knock down, break down, decrease or reduce (the levels or expression of) the targeted mutant allele.
  • an antisense oligonucleotide of the invention comprises 15 to 30 nucleotides including a central region of consecutive DNA nucleotides (and/or DNA nucleotide analogues) flanked at each end by wing regions comprising RNA nucleotides analogues.
  • an antisense oligonucleotide of the invention comprises 15 to 30 nucleotides including a central region of consecutive DNA nucleotides (and/or DNA nucleotide analogues) flanked at each end by wing regions comprising consecutive RNA nucleotides analogues.
  • an antisense oligonucleotide of the invention comprises 15 to 30 nucleotides including a central region of 5 to 15 consecutive DNA nucleotides (and/or DNA nucleotide analogues) flanked at each end by wing regions comprising 1 to 5 RNA nucleotide analogues.
  • an antisense oligonucleotide of the invention comprises 15 to 30 nucleotides including a central region of 5 to 15 consecutive DNA nucleotide analogues flanked in each end by wing regions comprising 1 to 5 RNA nucleotide analogues.
  • the antisense oligonucleotide comprising 15 to 30 nucleotides including a central region of (consecutive) DNA nucleotides (and/or DNA nucleotide analogues) flanked at each end by wing regions comprising (consecutive) RNA nucleotides analogues relates to the antisense oligonucleotide of the second aspect of the invention.
  • the antisense oligonucleotide comprising 15 to 30 nucleotides including a central region of 5 to 15 (consecutive) DNA nucleotides (and/or DNA nucleotide analogues) flanked at each end by wing regions comprising 1 to 5 RNA nucleotides analogues relates to the antisense oligonucleotide of the second aspect of the invention.
  • the central region comprises 5, 6, 7, 8, 9 , 10, 11 , 12, 13, 14 or 15 DNA nucleotides and/or DNA nucleotide analogues.
  • the length of the central region is 9, 10 or 11 DNA nucleotides and/or DNA nucleotide analogues.
  • one wing region comprises 1 , 2, 3, 4 or 5 RNA nucleotide analogues.
  • the length of one wing region is 2 or 4 RNA nucleotide analogues.
  • each wing region comprises 1 , 2, 3, 4 or 5 RNA nucleotide analogues.
  • the length of each wing region is 2 or 5 RNA nucleotide analogues. It is also encompassed by the invention that each wing region is not identical in length and/or in the type of chemistry used in the RNA nucleotide analogues.
  • the central region comprises 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 consecutive DNA nucleotides and/or DNA nucleotide analogues.
  • the length of the central region is 9, 10 or 11 consecutive DNA nucleotides and/or DNA nucleotide analogues.
  • one wing region comprises 1 , 2, 3, 4 or 5 consecutive RNA nucleotide analogues.
  • the length of one wing region is 2 or 5 consecutive RNA nucleotide analogues.
  • each wing region comprises 1 , 2, 3, 4 or 5 consecutive RNA nucleotide analogues.
  • each wing region is 2 or 5 consecutive RNA nucleotide analogues. It is also encompassed by the invention that each wing region is not identical in length and/or in the type chemistry used in the RNA nucleotide analogues.
  • the central region is 10 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 5 consecutive RNA nucleotide analogues. In another embodiment, the central region is 10 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 4 consecutive RNA nucleotide analogues. In another embodiment, the central region is 10 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 3 consecutive RNA nucleotide analogues.
  • the central region is 10 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 2 consecutive RNA nucleotide analogues. In another embodiment, the central region is 10 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 1 consecutive RNA nucleotide analogue.
  • the central region is 11 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 5 consecutive RNA nucleotide analogues, In another embodiment, the central region is 11 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 4 consecutive RNA nucleotide analogues, In another embodiment, the central region is 11 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 3 consecutive RNA nucleotide analogues, In another embodiment, the central region is 11 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 2 consecutive RNA nucleotide analogues, In another embodiment, the central region is 11 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 1 consecutive RNA nucleotide analogue.
  • the central region is 9 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 5 consecutive RNA nucleotide analogues. In another embodiment, the central region is 9 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 4 consecutive RNA nucleotide analogues. In another embodiment, the central region is 9 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 3 consecutive RNA nucleotide analogues.
  • the central region is 9 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 2 consecutive RNA nucleotide analogues. In another embodiment, the central region is 9 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 1 consecutive RNA nucleotide analogue.
  • the central region is 12 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 5 consecutive RNA nucleotide analogues. In another embodiment, the central region is 12 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 4 consecutive RNA nucleotide analogues. In another embodiment, the central region is 12 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 3 consecutive RNA nucleotide analogues.
  • the central region is 12 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 2 consecutive RNA nucleotide analogues. In another embodiment, the central region is 12 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 1 consecutive RNA nucleotide analogue.
  • the mutation in the allele or transcript of PSEN1 or PSEN2 is targeted by a nucleotide of the oligonucleotide being present in the central part of the oligonucleotide; the central part being 5 to 15 nucleotides and each wing being 1 to 5 nucleotides.
  • the antisense oligonucleotide comprising a nucleotide, present in the central part of the oligonucleotide, which targets the mutation in the allele or transcript of PSEN1 or PSEN2 relates to the antisense oligonucleotide of the second aspect of the invention.
  • the efficacy and selectivity of the oligonucleotide for the mutated PSEN1 or PSEN2 allele or transcript is optimal when the nucleotide of the oligonucleotide targeting the mutated nucleotide of PSEN1 or PSEN2 is located in the central part of the oligonucleotide.
  • antisense oligonucleotides have an increased selectivity towards the mutant PSEN1 allele when the nucleotide targeting the mutated nucleotide of PSEN1 is located within the first five nucleotides of the central part of the oligonucleotide starting from 5’ to 3’ (i.e;, nucleotides located at position 6, 7, 8, 9, or 10 in the oligonucleotide sequence starting from 5’ to 3’).
  • said nucleotide is the first, second, third, fourth, fifth, sixth, seventh, eighth, nineth, tenth, eleventh or twelfth nucleotide in the central part of the oligonucleotide starting from 5’ to 3’.
  • said nucleotide is the sixth, seventh, eighth, nineth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, or eighteenth nucleotide in the oligonucleotide sequence starting from 5’ to 3’.
  • said nucleotide is the first, second, third, fourth, or fifth nucleotide in the central part of the oligonucleotide starting from 5’ to 3’. Accordingly, in said preferred embodiment, said nucleotide is the sixth, seventh, eighth, nineth, or tenth nucleotide in the oligonucleotide sequence starting from 5’ to 3’. In an embodiment, said nucleotide is the first, second, third, or fifth nucleotide in the central part of the oligonucleotide starting from 5’ to 3’.
  • said nucleotide is the sixth, seventh, eight, or tenth nucleotide in the oligonucleotide sequence part starting from 5’ to 3’.
  • said base of said nucleotide present in the oligonucleotide and targeting the mutation is a thymine
  • said thymine is replaced by an uracil.
  • Examples are provided of oligonucleotides targeting the A431 E mutated allele of PSEN1 . Such examples are disclosed in SEQ ID NO: 26, 28, 30, 33, 35, 37, 39.
  • oligonucleotides targeting the S212Y mutated allele of PSEN1 Such examples are disclosed in SEQ ID NO: 188, 190, 192, 194, 198, 200.
  • Preferred oligonucleotides targeting this mutation via a thymine have this thymine be replaced by an uracil and this uracil is at the first, second or third position of their central part. Accordingly, preferred oligonucleotides targeting this mutation have the thymine replaced by an uracil at the sixth, seventh, or eight position of the oligonucleotide sequence starting from 5’ to 3’.
  • oligonucleotides targeting this mutation via a thymine have this thymine be replaced by an uracil and this uracil is at the first, second, third, or seventh position of the central part of the oligonucleotide.
  • Said preferred oligonucleotides have the thymine replaced by an uracil at the sixth, seventh, eighth, or twelfth position of the oligonucleotide sequence starting from 5’ to 3’. This replacement increases mutant allele selectivity, possibly due to a slight decreased Tm.
  • Preferred examples are found in SEQ ID NO: 28, 30, 33, 37, 39, 47, 97, 99, 115, 192, 194, 198.
  • the oligonucleotide is as follows: it comprises a central part of 5 to 15 nucleotides and two wings, each of 1 to 5 nucleotides and the mutation present in the mutated allele or transcript of PSEN1 or PSEN2 is targeted by the first, second, third, fourth, fifth, sixth, seventh, eighth, nineth, tenth, eleventh or twelfth nucleotide of the central part of the oligonucleotide.
  • the oligonucleotide is as follows: it comprises a central part of 5 to 15 nucleotides and two wings, each of 1 to 5 nucleotides and the mutation present in the mutated allele or transcript of PSEN1 or PSEN2 is targeted by the sixth, seventh, eighth, nineth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, or eighteenth nucleotide in the oligonucleotide sequence starting from 5’ to 3’.
  • the antisense oligonucleotide relates to the antisense oligonucleotide of the second aspect of the invention.
  • the base of the nucleotide of the oligonucleotide targeting the mutation present in the PSEN1 or PSEN2 allele or transcript is a RNA base.
  • said RNA base is a uracil.
  • the target mutation is a C to A mutations, but also the target mutation might be a G or T to A mutations.
  • the antisense oligonucleotide of the invention comprises nucleotides and nucleotide analogues.
  • Nucleotides may be RNA or DNA nucleotides.
  • the most common naturally occurring nucleotides in RNA are adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, thymidine monophosphate, and uridine monophosphate. These consist of a pentose sugar ribose, a 5’-linked phosphate group which is linked via a phosphate ester, and a T-linked base. The same holds for DNA wherein deoxyribose is present instead of ribose.
  • the sugar connects the base and the phosphate linkage, and is therefore often referred to as the scaffold of the nucleotide.
  • a modification in the pentose sugar is therefore often referred to as a scaffold modification.
  • a sugar modification may therefore be called a scaffold modification.
  • the original pentose sugar may be replaced in its entirety by another moiety that similarly connects the base and the phosphate. It is therefore understood that while a pentose sugar is often a scaffold, a scaffold is not necessarily a pentose sugar.
  • a base sometimes called a nucleobase, is generally adenine, cytosine, guanine, thymine, or uracil, or a derivative thereof. Cytosine, thymine, and uracil are pyrimidine bases, and are generally linked to the scaffold through their 1 -nitrogen. Adenine and guanine are purine bases, and are generally linked to the scaffold through their 9-nitrogen. A base present in an oligonucleotide is usually complementary to the target region. If such a base is a modified base or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • Base pairing refers to the binding of two bases (or nucleobases) to each other by hydrogen bonds. Specifically, a nucleobase analogue replacing cytosine is capable of base pairing with guanine, a nucleobase analogue replacing guanine is capable of base pairing with cytosine, a nucleobase analogue replacing adenine is capable of base pairing with uracil and a nucleobase replacing uracil is capable of base pairing with adenine.
  • a nucleotide is generally connected to neighboring nucleotides through condensation of its 5’- phosphate moiety to the 3’-hydroxyl moiety of the neighboring nucleotide monomer. Similarly, its 3’-hydroxyl moiety is generally connected to the 5’-phosphate of a neighboring nucleotide monomer. This forms phosphodiester bonds.
  • the phosphodiesters and the scaffold form an alternating copolymer. The bases are grafted to this copolymer, namely to the scaffold moieties. Because of this characteristic, the alternating copolymer formed by linked monomers of an oligonucleotide is often called the backbone of the oligonucleotide.
  • the phosphodiester bonds connect neighboring monomers together, they are often referred to as backbone linkages. It is understood that when a phosphate group is modified so that it is instead an analogous moiety such as a phosphorothioate (PS), such a moiety is still referred to as the backbone linkage of the monomer. This is referred to as a backbone linkage modification.
  • the backbone of an oligonucleotide is thus comprised of alternating scaffolds and backbone linkages.
  • the backbone of the oligonucleotide central part and the wings
  • is fully modified preferably being a PS backbone.
  • the antisense oligonucleotide comprises a central part comprising DNA nucleotides (or DNA nucleotides analogues) and modified RNA parts in each wing.
  • the antisense oligonucleotide comprising a central part comprising DNA nucleotides (or DNA nucleotides analogues) and modified RNA parts in each wing relates to the antisense oligonucleotide of the second aspect of the invention.
  • a RNA or DNA nucleotide may be modified and may therefore be considered as an RNA or DNA nucleotide analogue when it comprises a modified base and/or a modified sugar.
  • internucleoside linkage linking two adjacent nucleosides may be modified compared to the phosphodiester bond linking two nucleosides.
  • a “modified internucleoside linkage” may be replaced by the wording “backbone linkage modification” as explained earlier herein.
  • the antisense oligonucleotide of the invention comprises a DNA central part (or region) flanked at each end (wing) by modified RNA parts (regions). In another embodiment, the antisense oligonucleotide of the invention comprises a modified DNA central part (or region) flanked at each end (wing) by modified RNA parts (regions). In a preferred embodiment, the antisense oligonucleotide of the invention comprising a modified DNA central part flanked at each end by modified RNA parts relates to the antisense oligonucleotide of the second aspect of the invention.
  • the oligonucleotide of the invention consisting of a 5’ wing, a 3’ wing, and a central part (gap) is single-stranded.
  • Such structure a 5’ wing, a 3’ wing, and a central part (gap)
  • the single-stranded antisense oligonucleotide consisting of a 5’ wing, a 3’ wing, and a central part relates to the antisense oligonucleotide of the second aspect of the invention. Said structure is attractive as it allows the oligonucleotide to bind towards target mRNA and to recruit RNaseH.
  • the recruited RNaseH has at least one of the following effects on the targeted mutant allele: to silence, inactivate, knock down, break down, decrease or reduce its levels.
  • double-stranded siRNAs are not able to recruit RNaseH but rather depend on Argonaute proteins for silencing target mRNA.
  • Double-stranded siRNAs are loaded into the RNA-induced silencing complex (RISC). Upon RISC loading, the less thermodynamically stable 5'-end of the siRNA is incorporated and guides the RISC to the complementary target mRNA. The mRNA target dissociates from the intact siRNA after Argonaute-dependent cleavage, freeing RISC to regenerate and cleave additional mRNA targets.
  • RISC RNA-induced silencing complex
  • the presence of the wings provides the antisense oligonucleotide stability and resistance properties to degradation by exonucleases.
  • Implementation of chemical modifications in the gap and/or wings, comprising base, scaffold, and/or linkage modifications may improve safety, biodistribution, stability, cellular uptake, intracellular trafficking, target binding affinity, duplex stability and the efficiency of the oligonucleotide of the invention compared to an oligonucleotide consisting of non-modified DNA and/or non-modified RNA analogues. This effect may be at least due to the presence of the modified RNA wings and/or the modified DNA central part. Finetuning of DNA and/or RNA base, scaffold, and/or linkage modifications at specific positions in the oligonucleotide (precision chemistry) may result in oligonucleotides with most favorable characteristics for clinical application.
  • the central part/region (gap) of the antisense oligonucleotide comprises DNA nucleotides or DNA nucleotide analogues.
  • the base and sugar of this part of the antisense oligonucleotide are not modified.
  • an internucleoside linkage (or the whole backbone) in this central part is modified.
  • the modified internucleoside linkage is a phosphorothioate or a phosphoramidate internucleoside linkage.
  • a nucleotide of the central part/region of said antisense oligonucleotide may have at least one internucleoside linkage modification and/or at least one base modification compared to an antisense oligonucleotide with a full non-modified DNA central part.
  • a wing of the antisense oligonucleotide comprises modified RNA nucleotides and/or modified internucleoside linkages.
  • both wings of the antisense oligonucleotide comprise modified RNA nucleotides and/or modified internucleoside linkages.
  • a wing of said antisense oligonucleotide may have at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to a nonmodified RNA-based antisense oligonucleotide.
  • the modified internucleoside linkage is a phosphorothioate or a phosphoramidate internucleoside linkage.
  • a base modification in the central part and/or in the wing of the oligonucleotide includes a modified version of the natural purine and pyrimidine bases (e.g. adenine, uracil, guanine, cytosine, and thymine), such as hypoxanthine (e.g. inosine), orotic acid, agmatidine, lysidine, pseudouracil, 2- thiopyrimidine (e.g. 2-thiouracil, 2-thiothymine), G-clamp and its derivatives, 5-substituted pyrimidine (e.g.
  • adenine, uracil, guanine, cytosine, and thymine such as hypoxanthine (e.g. inosine), orotic acid, agmatidine, lysidine, pseudouracil, 2- thiopyrimidine (e.g. 2-thiouracil, 2-thiothymine), G-clamp and its derivatives
  • 5-halouracil 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5- hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, Super T), 7- deazaguanine, 7-deazaadenine, 2,6-diaminopurine, 7-aza-2,6-diaminopurine, 8-aza-7- deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, Super G, Super A, and N4-ethylcytosine, or derivatives thereof; N2-cyclopentylguanine (cPent-G), N2-cyclopentyl-2- aminopurine (cPent-AP), and N2-propyl-2-aminopurine (Pr-AP), or derivatives thereof; and degenerate or universal bases, like 2,6-difluorotoluene or absent bases like
  • a preferred modified base is 5-methylcytosine.
  • an antisense oligonucleotide of the invention may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 base modifications. It is also encompassed by the invention to introduce more than one distinct base modification in said wing of said antisense oligonucleotide.
  • an antisense oligonucleotide of the invention may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 base modifications. It is also encompassed by the invention to introduce more than one distinct base modification in the central part of said antisense oligonucleotide.
  • an antisense oligonucleotide of the invention may comprise at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more base modifications. It is also encompassed by the invention to introduce more than one distinct base modification in said antisense oligonucleotide.
  • a modified sugar in a nucleotide of a wing and/or in the central part of the antisense oligonucleotide is synonymous of a scaffold modification of the oligonucleotide.
  • a scaffold modification can include a modified version of the ribosyl moiety, such as 2’- O-modified RNA such as 2’-O-alkyl or 2’-O-(substituted)alkyl e.g. 2’-O-methyl, 2’-O-(2- cyanoethyl), 2’-0-(2-methoxy)ethyl (2’-MOE), 2’-0-(2-thiomethyl)ethyl, 2’-0-butyryl, 2’-O- propargyl, 2’-O-acetalester (such as e.g. Biscans et al. Bioorg. Med. Chem.
  • 2’-O-acetalester such as e.g. Biscans et al. Bioorg. Med. Chem.
  • a scaffold modification can include a bicyclic nucleic acid monomer (BNA) which may be a bridged nucleic acid monomer.
  • BNA bicyclic nucleic acid monomer
  • Each occurrence of said BNA may result in a monomer that is independently chosen from the group consisting of a conformationally restricted nucleotide (CRN) monomer, a locked nucleic acid (LNA) monomer, a xylo-LNA monomer, an a-LNA monomer, an a-L-LNA monomer, a p-D-LNA monomer, a 2’-amino-LNA monomer, a 2’-(alkylamino)-LNA monomer, a 2’-(acylamino)-LNA monomer, a 2’-/V-substituted-2’-amino-LNA monomer, a 2’-thio- LNA monomer, a (2’-O,4’-C) constrained eth
  • a preferred sugar modification is selected from: - 2’-0-modified RNA, more preferably 2’-0-alkyl or 2’-0-(substituted)alkyl, even more preferably 2’-O-methyl (2’-OMe) or 2’-0-(2-methoxy)ethyl (2’-MOE)
  • BNA BNA
  • CNN C-(CRN) monomer
  • LNA locked nucleic acid
  • an antisense oligonucleotide of the invention may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 sugar modifications. It is also encompassed by the invention to introduce more than one distinct sugar modification in said wing of said antisense oligonucleotide.
  • an antisense oligonucleotide of the invention may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 sugar modifications. It is also encompassed by the invention to introduce more than one distinct sugar modification in said wing of said antisense oligonucleotide.
  • an antisense oligonucleotide of the invention may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more sugar modifications. It is also encompassed by the invention to introduce more than one distinct sugar modification in said antisense oligonucleotide.
  • Antisense oligonucleotides according to the invention can comprise backbone linkage modifications in their wing and/or in their central part.
  • a backbone linkage modification can be, but is not limited to, a modified version of the phosphodiester present in RNA, such as phosphorothioate (PS), chirally pure phosphorothioate, (R)-phosphorothioate, (S)- phopshorothioate, phosphorodithioate (PS2), phosphonoacetate (PACE), phosphonoacetamide (PACA), thiophosphonoacetate (thioPACE), thiophosphonoacetamide, phosphorothioate prodrug, H-phosphonate, methyl phosphonate, methyl phosphonothioate, methyl phosphate, methyl phosphorothioate, ethyl phosphate, ethyl phosphorothioate, boranophosphate, boranophosphorot
  • Another modification includes phosphoryl guanidines, acylphosphoramidates, sulfonylphosphoramidates, phosphoramidite, phosphoramidate, N3’->P5’ phosphoramidate, phosphordiamidate, phosphorothiodiamidate, sulfamate, dimethylenesulfoxide, amide, sulfonate, siloxane, sulfide, sulfone, formacetyl, thioformacetyl, methylene formacetyl, alkenyl, methylenehydrazino, sulfonamide, amide, triazole, oxalyl, carbamate, methyleneimino (MMI), and thioacetamido nucleic acid (TANA); and their derivatives.
  • chirally pure phosphorothioate linkages are described in e.g. WO2014/010250 or WO2017/062862 (WaVe Life Sciences).
  • phosphoryl guanidine linkages are described in WO2016/028187 (Noogen).
  • Various salts, mixed salts and free acid forms are also included, as well as 3’->3’ and 2’->5’ linkages.
  • Preferred backbone linkage modifications are phosphorothioate and phosphoramidate.
  • an antisense oligonucleotide of the invention may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or more backbone linkage modifications. It is also encompassed by the invention to introduce more than one distinct backbone modification in said antisense oligonucleotide.
  • the antisense oligonucleotide is such that a DNA nucleotide in its central part is not modified.
  • the antisense oligonucleotide is such that a DNA nucleotide in its central part is modified and that at least part of the backbone (i.e. internucleoside linkage) in its central part comprises phosphorothioate and/or phosphoramidate (preferably a mesyl- phosphoramidate).
  • the antisense oligonucleotide is such that the RNA nucleotide analogues in each of its wings have been modified comprising a modified internucleoside linkage (preferably a phosphorothioate and/or phosphoramidite (preferably a mesyl-phosphoramidate)) and/or a modified sugar (preferably a 2’-MOE, 2’-OMe and/or a locked nucleic acid (LNA) monomer) and/or a modified base (preferably a 5-methylcytosine).
  • a modified internucleoside linkage preferably a phosphorothioate and/or phosphoramidite (preferably a mesyl-phosphoramidate)
  • a modified sugar preferably a 2’-MOE, 2’-OMe and/or a locked nucleic acid (LNA) monomer
  • a modified base preferably a 5-methylcytosine
  • the antisense oligonucleotide is such that:
  • RNA nucleotide analogues in each of its wings have been modified comprising a modified internucleoside linkage (preferably a phosphorothioate or phosphoramidate (more preferably a mesyl-phosphoramidate) and/or a modified sugar (preferably a 2’-MOE, 2’-OMe and/or a locked nucleic acid (LNA) monomer) and/or a modified base (preferably a 5-methylcytosine).
  • a modified internucleoside linkage preferably a phosphorothioate or phosphoramidate (more preferably a mesyl-phosphoramidate) and/or a modified sugar (preferably a 2’-MOE, 2’-OMe and/or a locked nucleic acid (LNA) monomer) and/or a modified base (preferably a 5-methylcytosine).
  • the antisense oligonucleotide is such that:
  • a DNA nucleotide in its central part has been modified and that the backbone (i.e. at least one internucleoside linkage) in its central comprises phosphorothioate or phosphoramidate (more preferably a mesyl-phosphoramidate) and
  • RNA nucleotide analogues in each of its wings have been modified comprising a modified internucleoside linkage (preferably a phosphorothioate or phosphoramidate (more preferably a mesyl-phosphoramidate) and/or a modified sugar (preferably a 2’-MOE, 2’-OMe and/or a locked nucleic acid (LNA) monomer) and/or a modified base (preferably a 5-methylcytosine).
  • a modified internucleoside linkage preferably a phosphorothioate or phosphoramidate (more preferably a mesyl-phosphoramidate) and/or a modified sugar (preferably a 2’-MOE, 2’-OMe and/or a locked nucleic acid (LNA) monomer) and/or a modified base (preferably a 5-methylcytosine).
  • the antisense oligonucleotide comprises 15 to 30 or 20 to 30 nucleotides including a central region of 5 to 15 consecutive DNA nucleotides flanked at each end by wing regions comprising 1 to 5 RNA nucleotides analogues.
  • the backbone of the oligonucleotide is fully modified (central region and each wing). More preferably the backbone is phosphorothioate and/or phosphoramidite (more preferably mesyl-phosphoramidate).
  • the antisense oligonucleotide is as follows:
  • RNA nucleotide analogues in each of its wings have been modified comprising a modified internucleoside linkage (preferably a phosphorothioate or phosphoramidite (more preferably mesyl-phosphoramidate)) and/or a modified sugar (preferably a 2’- MOE, 2’-OMe and/or a locked nucleic acid (LNA) monomer) and/or a modified base (preferably a 5-methylcytosine), or
  • a DNA nucleotide in its central part has been modified and that the backbone (i.e. at least one internucleoside linkage) in its central part comprises phosphorothioate and/or phosphoramidate (more preferably mesyl-phosphoramidate) and the RNA nucleotide analogues in each of its wings have been modified comprising a modified internucleoside linkage (preferably a phosphorothioate or phosphoramidate (more preferably mesyl-phosphoramidate) and/or a modified sugar (preferably a 2’- MOE, 2’-OMe and/or a locked nucleic acid (LNA) monomer) and/or a modified base (preferably a 5-methylcytosine).
  • a modified internucleoside linkage preferably a phosphorothioate or phosphoramidate (more preferably mesyl-phosphoramidate) and/or a modified sugar (preferably a 2’- MOE, 2’-OM
  • the antisense oligonucleotide is as follows:
  • RNA nucleotide analogues in each of its wings have been modified comprising a modified internucleoside linkage (preferably a phosphorothioateand/or phosphoramidite (more preferably mesyl-phosphoramidate)) and/or a modified sugar (preferably a 2’-MOE and/or a modified base (preferably a 5-methylcytosine), or
  • a DNA nucleotide in its central part has been modified and that the backbone (i.e. at least one internucleoside linkage) in its central part has been modified comprises a phosphorothioate and/or phosphoramidate (more preferably mesyl-phosphoramidate) and the RNA nucleotide analogues in each of its wings have been modified comprising a modified internucleoside linkage (preferably a phosphorothioate and/or phosphoramidite (more preferably mesyl-phosphoramidate)) and/or a modified sugar (preferably a 2’-MOE and/or a modified base (preferably a 5-methylcytosine).
  • a modified internucleoside linkage preferably a phosphorothioate and/or phosphoramidite (more preferably mesyl-phosphoramidate)
  • a modified sugar preferably a 2’-MOE and/or a modified base (preferably a 5-methylcytosine).
  • the antisense oligonucleotide is as follows: its DNA nucleotides in its central part have been modified and its backbone (i.e. at least one internucleoside linkage) in its central part comprises phosphorothioate linkages, and the RNA nucleotide analogues in each of its wings have been modified comprising a modified backbone which is phosphorothioate linkages, and preferably at least one modified sugar (preferably a 2’-MOE) and/or at least one modified base (preferably a 5-methylcytosine).
  • the invention provides human antisense oligonucleotides, which preferentially target a mutated allele of a human protein of the y-secretase pathway, preferably the human protein of the y-secretase pathway is PSEN1.
  • the antisense oligonucleotide targets, hybridizes, binds and/or is reverse complementary to a mutated allele of a human protein of the y- secretase pathway, preferably PSEN1.
  • the base sequence of the oligonucleotide comprises any of SEQ ID NO:1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,
  • oligonucleotides comprising one of these base sequences are disclosed below.
  • the antisense oligonucleotide comprises the following base sequence: TCTTTCTTGAAAATGGCAAG (AON1) (SEQ ID NO:1)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:1.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:1 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:1 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:1 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:1 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:1 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:1 is represented by SEQ ID NO:2: eT s eC* s eT s eT s eT s dC s dT s dT s dG s dA s dA s dA s dT s dG s eG s eC* s eA s eA s eG (SEQ ID NO:2) wherein A means adenosine, G means guanine, T means thymine, C means cytosine, C* means 5-methylcytosine, U means Uracil, e means 2’-MOE, d means DNA, subscript s means a PS internucleoside linkage, subscript o means PO linkage, subscript PNms means PNms linkage, and subscript PNdmi means PNdmi linkage.
  • the antisense oligonucleotide comprises the following base sequence: ATTCTTTCTTGAAAATGGCA (AON2) (SEQ ID NO:3)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:3.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:3 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:3 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:3 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:3 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:3 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:3 is represented by SEQ ID NO:4: eA s eT s eT s eC* s eT s dT s dT s dC s dT s dT s dG s dA s dA s dA s dA s eT s eG s eG s eC* s eA (SEQ ID NO:4)
  • the antisense oligonucleotide comprises the following base sequence: CAATTCTTTCTTGAAAATGG (AON3) (SEQ ID NO:5)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:5.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:5 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:5 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate) . More preferably, the oligonucleotide comprising SEQ ID NO:5 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:5 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:5 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:5 is represented by SEQ ID NO:6: eC* s eA s eA s eT s eT s dC s dT s dT s dT s dC s dT s dT s dG s dA s dA s eA s eA s eT s eG eG (SEQ ID NO:6)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:7.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:7 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:7 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NOT is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NOT has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:7 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:7 is represented by SEQ ID NO:8: eG s eC* s eA s eA s eT s dT s dC s dT s dT s dT s dC s dT s dT s dG (SEQ ID NO:8)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:9.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:9 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:9 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate) . More preferably, the oligonucleotide comprising SEQ ID NO:9 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:9 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:9 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:9 is represented by SEQ ID NQ:10: eG s eG s eC* s eA s eA s dT s dT s dC s dT s dT s dT s dC s dT s dT s dT s dG s eA s eA s eA s eT (SEQ ID NQ:10)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:11 .
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:11 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:11 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate) . More preferably, the oligonucleotide comprising SEQ ID NO:11 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:11 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:11 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:11 is represented by SEQ ID NO:12: eT s eG s eG s eC* s eA s dA s dT s dT s dC s dT s dT s dT s dC s dT s dT s eG s eA s eA s eA (SEQ ID NO:12)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:13.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO: 13 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:13 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate ). More preferably, the oligonucleotide comprising SEQ ID NO:13 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:13 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:13 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:13 is represented by SEQ ID NO:14:
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:15.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO: 15 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:15 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:15 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:15 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:15 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:15 is represented by SEQ ID NO:16: eA s eG s eC* s eT s eG s dG s dC s dA s dA s dT s dT s dC s dT s dT s dT s eC* s eT s eT s eG s eA (SEQ ID NO:16)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:17.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO: 17 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:17 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 17 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:17 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:17 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:17 is represented by SEQ ID NO:18: eA s eG s eA s eG s eC* s dT s dG s dG s dC s dA s dA s dT s dT s dC s dT s eT s eT s eC* s eT s eT (SEQ ID NO:18)
  • the antisense oligonucleotide comprises the following base sequence: GAAGAGCTGGCAATTCTTTC (AQN10) (SEQ ID NO:19)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:19.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO: 19 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:19 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate) . More preferably, the oligonucleotide comprising SEQ ID NO:19 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:19 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:19 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:19 is represented by SEQ ID NQ:20: eG s eA s eA s eG s eA s dG s dC s dT s dG s dG s dC s dA s dA s dT s dT s eC* s eT s eT s eT s eC*
  • the antisense oligonucleotide comprises the following base sequence: (AON11) TGGAAGAGCTGGCAATTCTT (SEQ ID NO:21)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:21 .
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:21 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:21 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate) . More preferably, the oligonucleotide comprising SEQ ID NO:21 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:21 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:21 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:21 is represented by SEQ ID NO:22:
  • the antisense oligonucleotide comprises the following base sequence: ATTGGAAGAGCTGGCAATTC (AON 12) (SEQ ID NO:23)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:23.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:23 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:23 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:23 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:23 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:23 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:23 is represented by SEQ ID NO:24: eA s eT s eT s eG s eG s dA s dA s dG s cLA s dG s dC s dT s dG s dG s dC s eA s eA s eT s eT s eC* (SEQ ID NO:24)
  • the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON13) (SEQ ID NO:25)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:25.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:25 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:25 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:25 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:25 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:25 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:25 is represented by SEQ ID NO:26: eG s eC* s eA s eA s eT s dU s dC s dT s dT s dT s dC s dT s dT s dG s dA s eA s eA s eT s eG (SEQ ID NO:26)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON14) (SEQ ID NO:27)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:27.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:27 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:27 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:27 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:27 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:27 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:27 is represented by SEQ ID NO:28: eG s eG s eC* s eA s eA s dT s dU s dC s dT s dT s dT s dC s dT s dT s dG s eA s eA s eA s eT (SEQ ID NO:28)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:29.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • at least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:29 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:29 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:29 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:29 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:29 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:29 is represented by SEQ ID NQ:30: eT s eG s eG s eC* s eA s dA s dT s dU s dC s dT s dT s dT s dC s dT s dT s eG s eA s eA s eA eA (SEQ ID NQ:30)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON16) (SEQ ID NO: 32)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:32.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:32 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:32 present in the gap of said oligonucleotide are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:32 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:32 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:32 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, , the oligonucleotide comprising SEQ ID NO:32 is fully modified with a PS backbone.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:32 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:32 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:32 is represented by SEQ ID NO:33: eGseGoeC*oeAoeAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdGseAoeAoeA o eAseT (SEQ ID NO:33)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON17) (SEQ ID NO: 34)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:34.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:34 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:32 present in the gap of said oligonucleotide (C at position 8 and at position 12 of SEQ ID NO:34) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:34 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:34 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:34 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NO:34 is fully modified with a PS backbone.
  • the oligonucleotide comprising SEQ ID NO:34 has at least one of its internucleoside linkages which is modified, preferably as a PNms linkage, preferably at least two of them.
  • a PNms linkage is present in the central region of the oligonucleotide and not in its wings.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:34 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:34 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:34 is represented by SEQ ID NO:35: eGseGseC*seAseAsdTsdUpNmsdCpNmsdTsdTsdTsdCsdTsdTsdTsdGseAseAseA s eT (SEQ ID NO:35)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON18) (SEQ ID NO: 36)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:36.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:36 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:36 present in the gap of said oligonucleotide are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:36 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:36 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:36 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NO:36 is fully modified with a PS backbone.
  • the oligonucleotide comprising SEQ ID NO:36 has at least one of its internucleoside linkages which is modified, preferably as a PNms linkage, preferably at least two of them.
  • a PNms linkage is present in the central region of the oligonucleotide and not in its wings.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:36 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:36 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:36 is represented by SEQ ID NO:37: eGseGseC*seAseAsdTsdUsdCpNmsdTpNmsdTsdTsdCsdTsdTsdGseAseAseAseT (SEQ ID NO:37)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON19) (SEQ ID NO: 38)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:38. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:38 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:38 present in the gap of said oligonucleotide are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:38 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:38 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:38 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO: 38 is fully modified with a PS backbone.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
  • the oligonucleotide comprising SEQ ID NO:38 has at least one of its internucleoside linkages which is modified, preferably as a PNms linkage, preferably at least two of them.
  • a PNms linkage is present in the central region of the oligonucleotide and not in its wings.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:38 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:38 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:38 is represented by SEQ ID NO:39: eGseGoeC*oeAoeAsdTsdUsdCpNmsdTpNmsdTsdTsdCsdTsdTsdGseAoeAoeAseT (SEQ ID NO:39)
  • the antisense oligonucleotide comprises the following base sequence: GGCAAATCTTTCTTGAAAAT (AON22) (SEQ ID NO: 40)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:40.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:40 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:40 present in the gap of said oligonucleotide are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NQ:40 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NQ:40 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NQ:40 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:40 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:40 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:40 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NQ:40 is represented by SEQ ID NO:41 : eGseGseC*seAseAsdAsdTsdCsdTsdTsdTsdTsdTsdGseAseAseA s eT (SEQ ID NO:41)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATTGTTTCTTGAAAAT (AON23) (SEQ ID NO: 42)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:42.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:42 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:42 present in the gap of said oligonucleotide (C at position 12 of SEQ ID NO:42) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:42 are 5- m ethyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:42 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:42 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:42 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:42 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:42 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:42 is represented by SEQ ID NO:43: eGseGseC*seAseAsdTsdTsdGsdTsdTsdTsdCsdTsdTsdGseAseAseA s eT (SEQ ID NO:43)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATTGATTCTTGAAAAT (AON24) (SEQ ID NO: 44)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:44.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:44 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:44 present in the gap of said oligonucleotide (C at position 12 of SEQ ID NO:44) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:44 are 5- methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:44 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:44 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:44 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:44 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:44 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:44 is represented by SEQ ID NO:45: eGseGseC*seAseAsdTsdTsdGsdAsdTsdTsdCsdTsdTsdGseAseAseA s eT (SEQ ID NO:45)
  • the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON25) (SEQ ID NO: 46)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:46.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:46 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:46 present in the gap of said oligonucleotide are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:46 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:46 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:46 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:46 is fully modified with a PS backbone.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:46 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:46 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:46 is represented by SEQ ID NO:47: eGseC*oeAoeAoeTsdUsdCsdTsdTsdTsdCsdTsdTsdGsdAseAoeAoeA o eTseG (SEQ ID NO:47)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATTCTAACTTGAAAAT (AON26) (SEQ ID NO: 48)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:48.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:48 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:48 present in the gap of said oligonucleotide (C at position 8 and at position 12 of SEQ ID NO:48) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:48 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:48 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:48 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:48 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:48 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:48 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:48 is represented by SEQ ID NO:49: eGseGseC*seAseAsdTsdTsdCsdTsdAsdAsdCsdTsdTsdGseAseAseA s eT (SEQ ID NO:49)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATTCTTAGTTGAAAAT (AON27) (SEQ ID NO: 50)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:50.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NQ:50 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NQ:50 present in the gap of said oligonucleotide (C at position 8 of SEQ ID NO:50) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:50 are 5- m ethyl cytosines.
  • the oligonucleotide comprising SEQ ID NQ:50 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NQ:50 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:50 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:50 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:50 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NQ:50 is represented by SEQ ID NO:51 : eGseGseC*seAseAsdTsdTsdCsdTsdTsdAsdGsdTsdTsdGseAseAseA s eT (SEQ ID NO:51)
  • the antisense oligonucleotide comprises the following base sequence: CAATTCTTTCTTGAAAAT (AON28) (SEQ ID NO: 52)
  • the antisense oligonucleotide has a length of 18 to 50 nucleotides and comprises the base sequence SEQ ID NO:52.
  • the length may be 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:52 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:52 present in the gap of said oligonucleotide (C at position 6 and at position 10 of SEQ ID NO:52) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:52 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:52 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:52 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:52 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:52 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:52 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 4 nucleotides. Most preferably both wings comprise 4 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:52 is represented by SEQ ID NO:53: eC*seAseAseTsdTsdCsdTsdTsdTsdTsdTsdGsdAseAseA s eT (SEQ ID NO:53)
  • the antisense oligonucleotide comprises the following base sequence: GCAATTCTTTCTTGAAAA (AON29) (SEQ ID NO: 54)
  • the antisense oligonucleotide has a length of 18 to 50 nucleotides and comprises the base sequence SEQ ID NO:54.
  • the length may be 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:54 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:54 present in the gap of said oligonucleotide are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:54 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:54 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:54 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:54 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:54 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:54 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 4 nucleotides. Most preferably both wings comprise 4 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:54 is represented by SEQ ID NO:55: eGseC*seAseAsdTsdTsdCsdTsdTsdTsdTsdTsdTsdGseAseAseA s eA (SEQ ID NO:55)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATTCTTTCTTGAAA (AON30) (SEQ ID NO: 56)
  • the antisense oligonucleotide has a length of 18 to 50 nucleotides and comprises the base sequence SEQ ID NO:56.
  • the length may be 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:56 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:56 present in the gap of said oligonucleotide (C at position 8 and at position 12 of SEQ ID NO:56) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:56 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:56 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:56 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:56 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:56 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:56 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise4 nucleotides. Most preferably both wings comprise 4 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:56 is represented by SEQ ID NO:57: eGseGseC*seAsdAsdTsdTsdCsdTsdTsdTsdTsdTsdTseGseAseA s eA (SEQ ID NO:57)
  • the antisense oligonucleotide comprises the following base sequence: AATTCTTTCTTGAAAA (AON31) (SEQ ID NO: 58)
  • the antisense oligonucleotide has a length of 16 to 50 nucleotides and comprises the base sequence SEQ ID NO:58.
  • the length may be 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • at least one C of the oligonucleotide comprising SEQ ID NO:58 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:58 present in the gap of said oligonucleotide (C at positions 5 and 9 of SEQ ID NO:58) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:58 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:58 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:58 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:58 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:58 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:58 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:58 is represented by SEQ ID NO:59: eAseAseTsdTsdCsdTsdTsdTsdCsdTsdTsdGsdAseAseAseA (SEQ ID NO:59)
  • the antisense oligonucleotide comprises the following base sequence: CAATTCTTTCTTGAAA (AON32) (SEQ ID NO: 60)
  • the antisense oligonucleotide has a length of 16 to 50 nucleotides and comprises the base sequence SEQ ID NO:60.
  • the length may be 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NQ:60 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NQ:60 present in the gap of said oligonucleotide (C at position 6 and position 10 of SEQ ID NQ:60) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NQ:60 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NQ:60 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NQ:60 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:60 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:60 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:60 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 3 nucleotides. Most preferably both wings comprises nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NQ:60 is represented by SEQ ID NO:61 : eC*seAseAsdTsdTsdCsdTsdTsdTsdTsdTsdGseAseA s eA (SEQ ID NO:61)
  • the antisense oligonucleotide comprises the following base sequence: GCAATTCTTTCTTGAA (AON33) (SEQ ID NO: 62)
  • the antisense oligonucleotide has a length of 16 to 50 nucleotides and comprises the base sequence SEQ ID NO:62.
  • the length may be 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:62 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:62 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO:62) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:62 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:62 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:62 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:62 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:62 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:62 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 3 nucleotides. Most preferably both wings comprise 3 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:62 is represented by SEQ ID NO:63: eGseC eAsdAsdTsdTsdCsdTsdTsdTsdTsdTseGseA s eA (SEQ ID NO:63)
  • the antisense oligonucleotide comprises the following base sequence: ATTCTTTCTTGAAAATGGCA (AON34) (SEQ ID NO: 64)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:64.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:64 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:64 present in the gap of said oligonucleotide (C at position 8 of SEQ ID NO:64) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:64 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:64 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:64 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:64 is fully modified with a PS backbone.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:64 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:64 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:64 is represented by SEQ ID NO:65: eAseT o eToeC*oeTsdTsdTsdCsdTsdTsdTsdGsdAsdAsdAsdAseToeGoeG o eC*seA (SEQ ID NO:65)
  • the antisense oligonucleotide comprises the following base sequence: ATTCTTTCTTGAAAATGGCA (AON35) (SEQ ID NO: 66)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:66.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:66 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:66 present in the gap of said oligonucleotide (C at position 8 of SEQ ID NO:66) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:66 are 5- m ethyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:66 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:66 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:66 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:66 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:66 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:66 is a dmi-Phosphoramidate (PNdmi) linkage.
  • PNdmi dmi-Phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:66 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:66 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
  • the oligonucleotide has two distinct internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
  • a most preferred oligonucleotide comprising SEQ ID NO:66 is represented by SEQ ID NO:67: eA s eT PNdmieT PNdmieC*PNdmieTsdTsdTsdCsdTsdTsdTsdGsdAsdAsdAsdAseT PNdmieGpNdmieGpNdmieC*seA (SEQ ID NO:67)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:68.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:68 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:68 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:68) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:68 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:68 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:68 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:68 is fully modified with a PS backbone.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:68 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:68 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:68 is represented by SEQ ID NO:69: eGseG o eC*oeAoeAsdTsdTsdCsdTsdTsdTsdTsdTsdGseAoeAoeA o eAseT (SEQ ID NO:69)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATTCTTTCTTGAAAAT (AON37) (SEQ ID NO: 70)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:70.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • at least one C of the oligonucleotide comprising SEQ ID NO:70 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:70 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NQ:70) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NQ:70 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NQ:70 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NQ:70 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:70 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NQ:70 is a dmi-Phosphoramidate (PNdmi) linkage.
  • PNdmi dmi-Phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NQ:70 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NQ:70 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
  • the oligonucleotide has two distinct internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:70 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:70 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NQ:70 is represented by SEQ ID NO:71 : eG s eGpNdmieC*PNdmieApNdmieAsdT sdT sdCsdT sdT sdT sdCsdT sdTsdGseApNdmieApNdmieAseT (SEQ ID NO:71)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATTCTTTCTTGAAAAT (AON38) (SEQ ID NO: 72)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:72.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:72 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:72 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:72) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:72 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:72 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:72 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:72 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:72 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:72 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
  • a most preferred oligonucleotide comprising SEQ ID NO:72 is represented by SEQ ID NO:73: eGseGoeC*seA o eAsdTsdTsdCsdTsdTsdTsdTsdTsdTsdGseAoeAseA o eAseT (SEQ ID NO:73)
  • the antisense oligonucleotide comprises the following base sequence: AGCTGGCAATTCTTTCTTGA (AON39) (SEQ ID NO: 74)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:74.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:74 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:74 present in the gap of said oligonucleotide (C at position 7 and position 12 of SEQ ID NO:74) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:74 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:74 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:74 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:74 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:74 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:74 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
  • a most preferred oligonucleotide comprising SEQ ID NO:74 is represented by SEQ ID NO:75: eAseG o eC*oeToeGsdGsdCsdAsdAsdTsdTsdCsdTsdTsdTseC*oeToeT o eGseA (SEQ ID NO:75)
  • the antisense oligonucleotide comprises the following base sequence: AGCTGGCAATTCTTTCTTGA (AQN40) (SEQ ID NO: 76)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:76.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:76 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:76 present in the gap of said oligonucleotide (C at position 7 and position 12 of SEQ ID NO:76) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:76 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:76 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:76 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:76 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:76 is a dmi-Phosphoramidate (PNdmi) linkage.
  • PNdmi dmi-Phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:76 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:76 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
  • the oligonucleotide has two distinct internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:76 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:76 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:76 is represented by SEQ ID NO:77: eA s eGpNdmieC*PNdmieT PNdmieGsdGsdCsdAsdAsdTsdTsdCsdTsdTsdTseC*PNdmieT PNdmieT PNdmieGseA (SEQ ID NO:77)
  • the antisense oligonucleotide comprises the following base sequence: AGCTGGCAATTCTTTCTTGA (AON41) (SEQ ID NO: 78)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:78.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:78 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:78 present in the gap of said oligonucleotide (C at position 7 and position 12 of SEQ ID NO:78) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:78 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:78 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:78 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:78 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:78 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:78 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
  • a most preferred oligonucleotide comprising SEQ ID NO:78 is represented by SEQ ID NO:79: eAseGoeC*seT o eGsdGsdCsdAsdAsdTsdTsdCsdTsdTsdTseC*oeTseT o eGseA (SEQ ID NO:79)
  • the antisense oligonucleotide comprises the following base sequence: GAAGAGCTGGCAATTCTTTC (AON42) (SEQ ID NO: 80)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NQ:80.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NQ:80 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NQ:80 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NQ:80) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NQ:80 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NQ:80 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NQ:80 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:80 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:80 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:80 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
  • a most preferred oligonucleotide comprising SEQ ID NO:80 is represented by SEQ ID NO:81 : eGseA o eAoeGoeAsdGsdCsdTsdGsdGsdCsdAsdAsdTsdTseC*oeToeT o eTseC* (SEQ ID NO:81)
  • the antisense oligonucleotide comprises the following base sequence: GAAGAGCTGGCAATTCTTTC (AON43) (SEQ ID NO: 82)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:82.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:82 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:82 present in the gap of said oligonucleotide (C at position 7 and position 1 1 of SEQ ID NO:82) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:82 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:82 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:82 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:82 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:82 is a dmi-Phosphoramidate (PNdmi) linkage.
  • PNdmi dmi-Phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:82 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:82 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
  • the oligonucleotide has two distinct internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:82 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:82 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:82 is represented by SEQ ID NO:83: eGseApNdmieApNdmieGpNdmieAsdGsdCsdT sdGsdGsdCsdAsdAsdT sdT seC*PNdmieT PNdmieT PNdmieT seC* (SEQ ID NO:83)
  • the antisense oligonucleotide comprises the following base sequence: TGGCAATUCTTTCTTGAAAA (AON44) (SEQ ID NO: 84)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:84.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:84 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:84 present in the gap of said oligonucleotide (C at position 9 and position 13 of SEQ ID NO:84) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:84 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:84 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:84 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:84 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:84 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:84 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
  • a most preferred oligonucleotide comprising SEQ ID NO:84 is represented by SEQ ID NO:85: eTseGoeGoeC*oeAsdAsdTsdUsdCsdTsdTsdTsdCsdTsdTseGoeAoeA o eAseA (SEQ ID NO:85)
  • the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON45) (SEQ ID NO: 86)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:86.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:86 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:86 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO:86) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:86 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:86 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:86 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:86 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:86 is a dmi-Phosphoramidate (PNdmi) linkage.
  • PNdmi dmi-Phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:86 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:86 comprises two distinct modified internucleoside linkages, preferably dmi-Phosphoramidate and phosphorothioate.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi- Phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:86 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:86 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:86 is represented by SEQ ID NO:87: eGseC*PNdmieA o eApNdmieT sdUsdCsdT sdTsdTsdCsdTsdTsdGsdAseAoeAoeT PNdmieG (SEQ I D
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON46) (SEQ ID NO: 88)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:88.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:88 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:88 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:88) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:88 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:88 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:88 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:88 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:88 is a dmi-Phosphoramidate (PNdmi) linkage.
  • PNdmi dmi-Phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:88 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:88 comprises two distinct modified internucleoside linkages, preferably dmi-Phosphoramidate and phosphorothioate.
  • the oligonucleotide hasthree distinct internucleoside linkages, preferably phosphodiester, dmi-Phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:88 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:88 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:88 is represented by SEQ ID NO:89: eGseGpNdmieC*oeApNdmieAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdGseAoeAoeApNdmieT (SEQ ID NO:89)
  • the antisense oligonucleotide comprises the following base sequence: TGGCAATUCTTTCTTGAAAA (AON47) (SEQ ID NO: 90)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:90.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NQ:90 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NQ:90 present in the gap of said oligonucleotide (C at position 9 and position 13 of SEQ ID NQ:90) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NQ:90 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NQ:90 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NQ:90 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:90 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NQ:90 is a dmi-Phosphoramidate (PNdmi) linkage.
  • PNdmi dmi-Phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:90 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:90 comprises two distinct modified internucleoside linkages, preferably dmi-Phosphoramidate and phosphorothioate.
  • the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi-Phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:90 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:90 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NQ:90 is represented by SEQ ID NO:91 : eTseGpNdmieGoeC*PNdmieAsdAsdTsdUsdCsdTsdTsdTsdCsdTsdTseGoeAoeAoeApNdmieA (SEQ ID NO:91)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAA (AON48) (SEQ ID NO: 92)
  • the antisense oligonucleotide has a length of 18 to 50 nucleotides and comprises the base sequence SEQ ID NO:92.
  • the length may be 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:92 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:92 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:92) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:92 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:92 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:92 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:92 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:92 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:92 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 4 nucleotides. Most preferably both wings comprise 4 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:92 is represented by SEQ ID NO:93: eGseGseC*seAsdAsdTsdUsdCsdTsdTsdTsdCsdTsdTseGseAseA s eA (SEQ ID NO:93)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAA (AON49) (SEQ ID NO: 94)
  • the antisense oligonucleotide has a length of 18 to 50 nucleotides and comprises the base sequence SEQ ID NO:94.
  • the length may be 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:94 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:94 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:94) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:94 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:94 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:94 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:94 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:94 is a dmi-Phosphoramidate (PNdmi) linkage.
  • PNdmi dmi-Phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:94 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 1 or 2 or 3, most preferably 1.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:94 comprises two distinct modified internucleoside linkages, preferably dmi-Phosphoramidate and phosphorothioate.
  • at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi- Phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:94 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:94 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 4 nucleotides. Most preferably both wings comprise 4 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:94 is represented by SEQ ID NO:95: eGseGpNdmieC*oeAsdAsdTsdUsdCsdTsdTsdTsdCsdTsdTseGoeAoeApNdmieA (SEQ ID NO:95)
  • the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AQN50) (SEQ ID NO: 96)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:96.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:96 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:96 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO:96) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:96 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:96 has at least one of its internucleoside linkages which is modified, preferably as a PNms linkage, preferably at least two of them.
  • a PNms linkage is present in the central region of the oligonucleotide and not in its wings.
  • at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
  • the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl-phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:96 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:96 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:96 is represented by SEQ ID NO:97: eGseC*oeAoeAoeTsdUpNmsdCpNmsdTsdTsdTsdCsdTsdTsdGsdAseAoeAoeTseG (SEQ ID NO:97)
  • the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON51) (SEQ ID NO: 98)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:98.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:98 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:98 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO:98) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:98 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:98 has at least one of its internucleoside linkages which is modified, preferably as a PNms linkage, preferably at least two of them.
  • a PNms linkage is present in the central region of the oligonucleotide and not in its wings.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:98 comprises one modified internucleoside linkage, preferably phosphorothioate.
  • the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl-phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:98 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:98 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:98 is represented by SEQ ID NO:99: eGseC*oeAoeAoeTsdUsdCpNmsdTpNmsdTsdTsdCsdTsdTsdGsdAseAoeAoeA o eTseG (SEQ ID NO:99)
  • the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON52) (SEQ ID NO: 100)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:100.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NQ:100 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NQ:100 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NQ:100) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NQ:100 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NQ:100 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 100 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:100 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings or in the central part of the oligonucleotide comprising SEQ ID NQ:100 is a mesyl- phosphoramidate (PNms) linkage.
  • PNms mesyl- phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NQ:100 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
  • the central part of the oligonucleotide comprising SEQ ID NQ:100 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NQ:100 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
  • 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NQ:100 and no PNms linkage is present in the wings of said oligonucleotide.
  • the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
  • At least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
  • the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl-phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID N0:100 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID N0:100 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NQ:100 is represented by SEQ ID NQ:101 : eGseC*PNmseApNmseA o eT sd UsdCpNmsdT PNmsdT sdT sdCsdTsdT sdGsd AseAoeAoeAoeT seG (SEQ I D NQ:101)
  • the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON53) (SEQ ID NO: 102)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:102.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NQ:102 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NQ:102 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NQ:102) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NQ:102 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NQ:102 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 102 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:102 is fully modified with a PS backbone.
  • the oligonucleotide comprising SEQ ID NQ:102 has at least one of its internucleoside linkages which is modified, preferably as a PNms linkage, preferably at least two of them.
  • a PNms linkage is present in the central region of the oligonucleotide and not in one wing (or in both wings).
  • the oligonucleotide comprising SEQ ID NQ:102 has at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
  • the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl-phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:102 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:102 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NQ:102 is represented by SEQ ID NQ:103: eGseC*oeAoeAoeTsdUsdCpNmsdTpNmsdTsdTsdCsdTsdTsdGsdAseAseAseTseG (SEQ ID NQ:103)
  • the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON54) (SEQ ID NO: 104)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:104.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NQ:104 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NQ:104 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO: 104) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NQ:104 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NQ:104 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 104 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:104 is fully modified with a PS backbone.
  • the oligonucleotide comprising SEQ ID NQ:104 has at least one of its internucleoside linkages which is modified, preferably as a PNms linkage, preferably at least 2, 3, 4, 5, of them. In an embodiment, a PNms linkage is present in the central region of the oligonucleotide and not in its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:104 has at least one internucleoside linkages in one wing (or in both wings) which is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
  • the oligonucleotide has distinct internucleoside linkages, preferably phosphodiester, mesyl-phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:104 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:104 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NQ:104 is represented by SEQ ID NQ:105: eGseC*seAseAseTsdUsdCpNmsdTpNmsdTsdTsdCsdTsdTsdGsdAseAoeAoeTseG (SEQ ID NQ:105)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON55) (SEQ ID NO: 106)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:106.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NQ:106 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NQ:106 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NQ:106) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NQ:106 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NQ:106 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 106 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:106 is fully modified with a PS backbone.
  • the oligonucleotide comprising SEQ ID NQ:106 has at least one of its internucleoside linkages which is modified, preferably as a PNms linkage, preferably at least 2, 3, 4, 5 of them.
  • a PNms linkage is present in the central region of the oligonucleotide and not in one of its wings (or not in both wings).
  • the oligonucleotide comprising SEQ ID NO:106 has at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
  • the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl-phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:106 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:106 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NQ:106 is represented by SEQ ID NQ:107: eGseGoeC*oeAoeAsdTsdUpNmsdCpNmsdTsdTsdTsdCsdTsdTsdTsdGseAoeAoeAseT (SEQ ID NQ:107)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON56) (SEQ ID NO: 108)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:108.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NQ:108 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NQ:108 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO: 108) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NQ:108 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NQ:108 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 108 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:108 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings or in the central part of the oligonucleotide comprising SEQ ID NQ:108 is a mesyl- phosphoramidate (PNms) linkage.
  • PNms mesyl- phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:108 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
  • the central part of the oligonucleotide comprising SEQ ID NO:108 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NQ:108 comprises two distinct modified internucleoside linkages, preferably mesyl-phosphoramidate and phosphorothioate.
  • 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NQ:108 and no PNms linkage is present in the wings of said oligonucleotide.
  • the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:108 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:108 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NQ:108 is represented by SEQ ID NQ:109: eGseGpNmseC*PNmseA o eAsdT sdUsdCpNmsdT PNmsdT sdT sdCsdT sdT sdGseAoeAoeAseT (SEQ ID NQ:109)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON57) (SEQ ID NO: 110)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:110.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NQ:110 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NQ:110 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NQ:110) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:110 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:110 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:110 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:110 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NQ:110 is a mesyl-phosphoramidate (PNms) linkage.
  • PNms mesyl-phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:1 10 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NQ:1 10 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
  • 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NQ:110 and no PNms linkage is present in the wings of said oligonucleotide.
  • the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
  • the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester .mesyl-phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:110 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:110 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:110 is represented by SEQ ID NO:1 11 : eGseGpNmseC*PNmseAoeAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdTsdGseAoeAoeAseT (SEQ ID NO:111)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON58) (SEQ ID NO: 112)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:112.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:112 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:112 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO: 112) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:112 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:112 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:112 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:112 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:112 is a mesyl-phosphoramidate (PNms) linkage.
  • PNms mesyl-phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:1 12 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:1 12 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
  • 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:112 and no PNms linkage is present in the wings of said oligonucleotide.
  • the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:112 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:112 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:112 is represented by SEQ ID NO:1 13: eGseGoeC*PNmseApNmseAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdTsdGseAoeAoeAseT (SEO ID NO:113)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON59) (SEQ ID NO: 114)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:114.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:114 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:114 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO: 114) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:114 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:114 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:114 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:1 14 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:114 is a mesyl-phosphoramidate (PNms) linkage.
  • PNms mesyl-phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:114 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:114 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
  • 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:114 and no PNms linkage is present in the wings of said oligonucleotide.
  • the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
  • at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
  • the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl-phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:114 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:114 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:114 is represented by SEQ ID NO:1 15: eGseGoeC*oeAoeAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdGseAoeAoeApNmseApNmseT (SEQ ID NO:1 15)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AQN60) (SEQ ID NO: 116)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:116.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:116 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:116 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO: 116) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:116 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:116 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:116 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:116 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:116 is a mesyl-phosphoramidate (PNms) linkage.
  • PNms mesyl-phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:1 16 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:1 16 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
  • 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:116 and no PNms linkage is present in the wings of said oligonucleotide.
  • the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:116 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:116 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:116 is represented by SEQ ID NO:117: eGseGoeC*oeAoeAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdGseAoeApNmseApNmseAoeT (SEQ ID NO:117).
  • the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON61) (SEQ ID NO: 121)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:121 .
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:121 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:121 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO: 121) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:121 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:121 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:121 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:121 is fully modified with a PS backbone.
  • At least one sugar modification is present, preferably at least one 2-Omethyl sugar modification is present in the gap of said oligonucleotide. Most preferably, at position 7 of SEQ ID NO: 121 , a 2-Omethyl sugar modification is present.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:121 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:121 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:121 is represented by SEQ ID NO:122: eGseC*oeAoeAoeTsdUsmCsdTsdTsdTsdCsdTsdTsdTsdGsdAseAoeAoeA o eTseG (SEQ ID NO:122)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON62) (SEQ ID NO: 123)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:123.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:123 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:123 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:123) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:123 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:123 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 123 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:123 is fully modified with a PS backbone.
  • At least one sugar modification is present, preferably at least one 2-Omethyl sugar modification is present in the gap of said oligonucleotide. Most preferably, at position 7 of SEQ ID NO: 123, a 2-Omethyl sugar modification is present.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:123 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:123 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:123 is represented by SEQ ID NO:124: eGseGoeC*oeAoeAsdTsmUsdCsdTsdTsdTsdCsdTsdTsdGseAoeAoeA o eAseT (SEQ ID NO:124)
  • the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON63) (SEQ ID NO: 125)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:125.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:125 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:125 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO:125) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:125 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:125 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:125 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:125 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:125 is a dmi-Phosphoramidate (PNdmi) linkage.
  • PNdmi dmi-Phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO: 125 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 1 or 2 or 3, most preferably 1
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO: 125 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi- Phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:125 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:125 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:125 is represented by SEQ ID NO:126: eGseC*oeAoeAoeTsdUsdCsdTsdTsdTsdCsdTsdTsdGsdAseAoeAoeTpNdmieG (SEQ ID NO:126)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON64) (SEQ ID NO: 127)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:127.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:127 present in the gap of said oligonucleotide are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:127 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:127 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 127 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:127 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:127 is a dmi-Phosphoramidate (PNdmi) linkage.
  • PNdmi dmi-Phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:127 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 1 or 2 or 3, most preferably 1 .
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:127 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi- Phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:127 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:127 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:127 is represented by SEQ ID NO:128: eGseGoeC*oeAoeAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdGseAoeAoeApNdmieT (SEQ ID NO:128)
  • the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON65) (SEQ ID NO: 129)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:129.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:129 present in the gap of said oligonucleotide are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:129 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:129 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate).
  • the oligonucleotide comprising SEQ ID NO: 129 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings.
  • the oligonucleotide comprising SEQ ID NO:129 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:129 is a mesyl-phosphoramidate (PNms) linkage.
  • PNms mesyl-phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:129 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO: 129 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
  • 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:129 and no PNms linkage is present in the wings of said oligonucleotide.
  • the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
  • At least one sugar modification is present, preferably at least one 2-Omethyl sugar modification is present in the gap of said oligonucleotide. Most preferably, at position 7 of SEQ ID NO: 129, a 2-Omethyl sugar modification is present.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:129 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:129 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:129 is represented by SEQ ID NQ:130: eGseC*oeAoeAoeTsdUpNmsmCpNmsdTsdTsdTsdCsdTsdTsdTsdGsdAseAoeAoeTseG (SEQ ID NQ:130)
  • the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON66) (SEQ ID NO: 131)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:131 .
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:131 present in the gap of said oligonucleotide are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:131 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:131 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 131 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:131 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:131 is a mesyl-phosphoramidate (PNms) linkage.
  • PNms mesyl-phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:131 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:131 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
  • 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:131 and no PNms linkage is present in the wings of said oligonucleotide.
  • the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
  • at least one sugar modification is present, preferably at least one 2-Omethyl sugar modification is present in the gap of said oligonucleotide. Most preferably, at position 7 of SEQ ID NO: 131 , a 2-Omethyl sugar modification is present.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:131 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:131 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:131 is represented by SEQ ID NO:132: eGseC*oeAoeAoeTsdUsmCpNmsdTpNmsdTsdTsdCsdTsdTsdGsdAseAoeAoeTseG (SEQ ID NO:132)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON67) (SEQ ID NO: 133)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:133.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:133 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:133 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:133) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:133 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:133 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:133 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:133 is a mesyl-phosphoramidate (PNms) linkage.
  • PNms mesyl-phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:133 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:133 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
  • 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:133 and no PNms linkage is present in the wings of said oligonucleotide.
  • the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:133 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:133 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:133 is represented by SEQ ID NO:134: eGseGoeC*oeAoeAsdTsmUpNmsdCpNmsdTsdTsdTsdCsdTsdTsdTsdGseAoeAoeAseT (SEQ ID NO:134)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON68) (SEQ ID NO: 135)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:135.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:135 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:135 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:135) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:135 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:135 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 135 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:135 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:135 is a mesyl-phosphoramidate (PNms) linkage.
  • PNms mesyl-phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:135 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:135 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
  • 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:135 and no PNms linkage is present in the wings of said oligonucleotide.
  • the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:135 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:135 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • At least one sugar modification is present, preferably at least one 2-Omethyl sugar modification is present in the gap of said oligonucleotide. Most preferably, at position 7 of SEQ ID NO: 135, a 2-Omethyl sugar modification is present.
  • a most preferred oligonucleotide comprising SEQ ID NO:135 is represented by SEQ ID NO:136: eGseGoeC*oeAoeAsdTsmUsdCpNmsdTpNmsdTsdTsdCsdTsdTsdGseAoeAoeAseT (SEQ ID NO:136)
  • the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON69) (SEQ ID NO: 137)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:137.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:137 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:137 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO: 137) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:137 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:137 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 137 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:137 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:137 is a mesyl-phosphoramidate (PNms) linkage.
  • PNms mesyl-phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO: 137 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO: 137 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
  • 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:137 and no PNms linkage is present in the wings of said oligonucleotide.
  • the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:137 is a dmi-Phosphoramidate (PNdmi) linkage.
  • PNdmi dmi-Phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:137 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 1 or 2 or 3, most preferably 1 .
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:137 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:137 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:137 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:137 is represented by SEQ ID NO:138: eGseC*oeA o eA o eT sdUpNmsdCpNmsdT sdT sdTsdCsdTsdTsdTsdGsdAseAoeAoeT PNdmieG (SEQ I D NO:138)
  • the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AQN70) (SEQ ID NO: 139)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:139.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:139 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:139 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO:139) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:139 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:139 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 139 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:139 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:139 is fully modified with a PS backbone.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:139 is a mesyl-phosphoramidate (PNms) linkage.
  • PNms mesyl-phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:139 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:139 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
  • 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:139 and no PNms linkage is present in the wings of said oligonucleotide.
  • the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:139 is a dmi-Phosphoramidate (PNdmi) linkage.
  • PNdmi dmi-Phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:139 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 1 or 2 or 3, most preferably 1 .
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:139 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:139 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:139 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:139 is represented by SEQ ID NO:140: eGseC*oeA o eA o eT sdUsdCpNmsdT PNmsdT sdT sdCsdTsdTsdGsdAseAoeAoeT PNdmieG (SEO I D NO:140)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON71) (SEQ ID NO: 141)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:141 .
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:141 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:141 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:141) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:141 are 5- methyl cytosines.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:141 is a mesyl-phosphoramidate (PNms) linkage.
  • PNms mesyl-phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:141 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:141 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
  • 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:141 and no PNms linkage is present in the wings of said oligonucleotide.
  • the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:141 is a dmi-Phosphoramidate (PNdmi) linkage.
  • PNdmi dmi-Phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:141 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 1 or 2 or 3, most preferably 1
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:141 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
  • the oligonucleotide comprising SEQ ID NO:141 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 141 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:141 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:141 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:141 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:141 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:141 is represented by SEQ ID NO:142: eGseGoeC*oeA o eAsdT sdUpNmsdCpNmsdT sdT sdTsdCsdTsdT sdGseAoeAoeApNdmieT (SEQ ID NO:142)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON72) (SEQ ID NO: 143)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:143.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:143 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:143 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:143) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:143 are 5- methyl cytosines.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:143 is a mesyl-phosphoramidate (PNms) linkage.
  • PNms mesyl-phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:143 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:143 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
  • 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:143 and no PNms linkage is present in the wings of said oligonucleotide.
  • the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
  • one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:143 is a dmi-Phosphoramidate (PNdmi) linkage.
  • PNdmi dmi-Phosphoramidate
  • one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:143 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 1 or 2 or 3, most preferably 1 .
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:143 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
  • the oligonucleotide comprising SEQ ID NO:143 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 143 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:143 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:143 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:143 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:143 is represented by SEQ ID NO:144: eGseGoeC*oeA o eAsdT sdUsdCpNmsdT PNmsdT sdT sdCsdTsdT sdGseAoeAoeApNdmieT (SEQ ID NO:144)
  • the antisense oligonucleotide comprises the following base sequence: GTAAATCATTCCCACCACAC (AON73) (SEQ ID NO: 173)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:173.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:173 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:173 present in the gap of said oligonucleotide (C at positions 7, 11 , 12, 13, 15 of SEQ ID NO:173) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:173 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:173 has at least one of its internucleoside linkages which is modified, preferably as a phosphoroth ioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:173 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:173 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:173 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:173 is represented by SEQ ID NO:174: eGseTseAseAseAsdTsdCsdAsdTsdTsdCsdCsdCsdAsdCseC*seAseC*seA s eC* (SEQ ID NO:174).
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:175.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:175 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:175 present in the gap of said oligonucleotide (C at positions 9, 13-15 of SEQ ID NO: 175) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:175 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:175 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:175 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:175 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:175 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:175 is represented by SEQ ID NO:176: eAseTseGseTseAsdAsdAsdTsdCsdAsdTsdTsdCsdCseAseC*seC*seAseC* (SEQ ID NO:176).
  • the antisense oligonucleotide comprises the following base sequence: GAATGTAAATCATTCCCACC (AON75) (SEQ ID NO: 177)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:177.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:177 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:177 present in the gap of said oligonucleotide (C at position 11 and position 15 of SEQ ID NO:177) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO: 177 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:177 has at least one of its internucleoside linkages which is modified, preferably as a phosphoroth ioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:177 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:177 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:177 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:177 is represented by SEQ ID NO:178: eGseAseAseTseGsdTsdAsdAsdAsdTsdCsdAsdTsdTsdCseC*seC*seAseC* s eC* (SEQ ID NO:178)
  • the antisense oligonucleotide comprises the following base sequence: GTGAATGTAAATCATTCCCA (AON76) (SEQ ID NO: 179).
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:179.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:179 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:179present in the gap of said oligonucleotide (C at position 13 of SEQ ID NO:179) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:179 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:179 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:179 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:179 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:179 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:179 is represented by SEQ ID NQ:180: eGseTseGseAseAsdTsdGsdTsdAsdAsdTsdCsdAsdTseTseC*seC*seC* s eA (SEQ ID NO:180)
  • the antisense oligonucleotide comprises the following base sequence: CAGTGAATGTAAATCATTCC (AON77) (SEQ ID NO: 181).
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:181 .
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:181 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:181 present in the gap of said oligonucleotide (C at position 15 of SEQ ID NO:181) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:181 are 5- m ethyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:181 has at least one of its internucleoside linkages which is modified, preferably as a phosphoroth ioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 181 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:181 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:181 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:181 is represented by SEQ ID NO:182: eC*seAseGseTseGsdAsdAsdTsdGsdTsdAsdAsdTsdCseAseTseTseC* s eC* (SEQ ID NO:182)
  • the antisense oligonucleotide comprises the following base sequence: TCCAGTGAATGTAAATCATT (AON78) (SEQ ID NO: 183).
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:183.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • at least one C of the oligonucleotide comprising SEQ ID NO:183 is 5- methylcytosine. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:183 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:183 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:183 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:183 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:183 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:183 is represented by SEQ ID NO:184: eTseC*seC*seAseGsdTsdGsdAsdAsdTsdGsdTsdAsdAsdAseTseC*seAseT s eT (SEQ ID NO:184)
  • the antisense oligonucleotide comprises the following base sequence: TTTCCAGTGAATGTAAATCA (AON79) (SEQ ID NO: 185).
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:185.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:185 is 5- methylcytosine. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:185 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:185 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:185 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:185 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:185 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:185 is represented by SEQ ID NO:186: eTseTseTseC*seC*sdAsdGsdTsdGsdAsdAsdTsdGsdTsdAseAseTseC* s eA (SEQ ID NO:186)
  • the antisense oligonucleotide comprises the following base sequence: GUAAATCATTCCCACCACAC (AON 80) (SEQ ID NO: 187).
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:187.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:187 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:187 present in the gap of said oligonucleotide are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:187 are 5- methyl cytosines.
  • the oligonucleotide comprising SEQ ID NO:187 has at least one of its internucleoside linkages which is modified, preferably as a phosphoroth ioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:187 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:187 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:187 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:187 is represented by SEQ ID NO:188: eGseUseAseAseAsdTsdCsdAsdTsdTsdCsdCsdCsdAsdCseC*seAseC*seA s eC* (SEQ ID NO:188)
  • the antisense oligonucleotide comprises the following base sequence: ATGUAAATCATTCCCACCAC (AON 81 (SEQ ID NO: 189).
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:189.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • at least one C of the oligonucleotide comprising SEQ ID NO:189 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:189 present in the gap of said oligonucleotide (C at positions 9, 13-15, of SEQ ID NO:189) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:189 are 5- methyl cytosines.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:189 comprises one distinct modified internucleoside linkages, preferably phosphorothioate.
  • the oligonucleotide comprising SEQ ID NO:189 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:189 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:189 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:189 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:189 is represented by SEQ ID NQ:190: eAseTseGseUseAsdAsdAsdTsdCsdAsdTsdTsdCsdCseAseC*seC*seA s eC* (SEQ ID NQ:190)
  • the antisense oligonucleotide comprises the following base sequence: GAATGUAAATCATTCCCACC (AON 82 (SEQ ID NO: 191).
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:191 .
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:191 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:191 present in the gap of said oligonucleotide (C at position 11 and at position 15 of SEQ ID NO:191) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:191 are 5-methylcytosines.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:191 comprises one distinct modified internucleoside linkages, preferably phosphorothioate.
  • the oligonucleotide comprising SEQ ID NO:191 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:191 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:191 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:191 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:191 is represented by SEQ ID NO:192: eGseAseAseTseGsdUsdAsdAsdAsdTsdCsdAsdTsdTsdCseC*seC*seAseC* s eC* (SEQ ID NO:192)
  • the antisense oligonucleotide comprises the following base sequence: GTGAATGUAAATCATTCCCA (AON 83 (SEQ ID NO: 193).
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:193.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:193 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:193 present in the gap of said oligonucleotide (C at position 13 of SEQ ID NO:193) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:193 are 5- m ethyl cytosines.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:193 comprises one distinct modified internucleoside linkages, preferably phosphorothioate.
  • the oligonucleotide comprising SEQ ID NO:193 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:193 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:193 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:193 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:193 is represented by SEQ ID NO:194: eGseTseGseAseAsdTsdGsdUsdAsdAsdAsdTsdCsdAsdTseTseC*seC*seC* s eA (SEQ ID NO:194)
  • the antisense oligonucleotide comprises the following base sequence: CAGTGAATGUAAATCATTCC (AON 84 (SEQ ID NO: 195).
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:195.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:195 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:195 present in the gap of said oligonucleotide (C at position 15 of SEQ ID NO:195) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:195 are 5- m ethyl cytosines.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:195 comprises one distinct modified internucleoside linkages, preferably phosphorothioate.
  • the oligonucleotide comprising SEQ ID NO:195 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:195 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:195 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:195 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:195 is represented by SEQ ID NO:196: eC*seAseGseTseGsdAsdAsdTsdGsdUsdAsdAsdAsdTsdCseAseTseTseC* s eC* (SEQ ID NO:196)
  • the antisense oligonucleotide comprises the following base sequence: TCCAGTGAATGUAAATCATT (AON 85 (SEQ ID NO: 197).
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:197.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:197 is 5- methylcytosine. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:197 are 5- methyl cytosines.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:197 comprises one distinct modified internucleoside linkages, preferably phosphorothioate.
  • the oligonucleotide comprising SEQ ID NO:197 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:197 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:197 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:197 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:197 is represented by SEQ ID NO:198: eTseC*seC*seAseGsdTsdGsdAsdAsdTsdGsdUsdAsdAsdAseTseC*seAseT s eT (SEQ ID NO:198)
  • the antisense oligonucleotide comprises the following base sequence: TTTCCAGTGAATGUAAATCA (AON 86 (SEQ ID NO: 199).
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:199.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:199 is 5- methylcytosine. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:199 are 5- methyl cytosines.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:199 comprises one distinct modified internucleoside linkages, preferably phosphorothioate.
  • the oligonucleotide comprising SEQ ID NO:199 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:199 is fully modified with a PS backbone. In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:199 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:199 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:199 is represented by SEQ ID NQ:200: eTseTseTseC*seC*sdAsdGsdTsdGsdAsdAsdTsdGsdUsdAseAseTseC* s eA (SEQ ID NQ:200)
  • the antisense oligonucleotide comprises the following base sequence: AGCGCCGGCAATTCTTTCTTG (AON 88 (SEQ ID NO: 203).
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:203.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NQ:203 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NQ:203 present in the gap of said oligonucleotide (C at positions 6, 9, 14 of SEQ ID NQ:203) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NQ:203 are 5- methyl cytosines.
  • one wing or preferably each wing of the oligonucleotide comprising SEQ ID NQ:203 comprises one distinct modified internucleoside linkages, preferably phosphorothioate.
  • the oligonucleotide comprising SEQ ID NQ:203 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NQ:203 is fully modified with a PS backbone.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:203 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:203 comprises 1 , 2, 3, 4 or 5 nucleotides.
  • one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NQ:203 is represented by SEQ ID NQ:204: eAseG o eC*oeGoeC*sdCsdGsdGsdCsdAsdAsdTsdTsdCsdTsdTseToeC*oeT o eTseG (SEQ ID NQ:204)
  • the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON89) (SEQ ID NO: 218)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:218.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:218 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:218 present in the gap of said oligonucleotide (C at position 7 and at position 11 of SEQ ID NO:218) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:218 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:218 has at least one of its internucleoside linkages which is modified, preferably as a phosphoroth ioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:218 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NO:218 is fully modified with a PS backbone.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:218 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:218 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:218 is represented by SEQ ID NO:219: eGseC*oeAseAoeTsdUsdCsdTsdTsdTsdCsdTsdTsdGsdAseAoeAseA o eTseG (SEQ ID NO:219)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AQN90) (SEQ ID NO: 220)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:220.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:220 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:220 present in the gap of said oligonucleotide (C at position 8 and at position 12 of SEQ ID NQ:220) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NQ:220 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NQ:220 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate).
  • the oligonucleotide comprising SEQ ID NQ:220 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings.
  • the oligonucleotide comprising SEQ ID NQ:220 is fully modified with a PS backbone.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:220 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:220 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NQ:220 is represented by SEQ ID NO:221 : eGseGoeC*seAoeAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdGseAoeAseA o eAseT (SEQ ID NO:221)
  • the antisense oligonucleotide comprises the following base sequence: TGGCAATUCTTTCTTGAAAA (AON91) (SEQ ID NO: 222)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:222.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:222 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:222 present in the gap of said oligonucleotide (C at position 9 and at position 13 of SEQ ID NO:222) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:222 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:222 has at least one of its internucleoside linkages which is modified, preferably as a phosphoroth ioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:222 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NO:222 is fully modified with a PS backbone.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:222 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:222 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:222 is represented by SEQ ID NO:223: eTseGoeGseC*oeAsdAsdTsdUsdCsdTsdTsdTsdCsdTsdTseGoeAseA o eAseA (SEQ ID NO:223)
  • the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON92) (SEQ ID NO: 224)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:224.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:224 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:224 present in the gap of said oligonucleotide are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:224 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:224 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:224 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NO:224 is fully modified with a PS backbone.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:224 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:224 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:224 is represented by SEQ ID NO:225: eGseC*seAseAseTsdUsdC*sdTsdTsdTsdC*sdTsdTsdGsdAseAseAseT s eG (SEQ ID NO:225)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON93) (SEQ ID NO: 226)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:226.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:226 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:226 present in the gap of said oligonucleotide (C at position 8 and at position 12 of SEQ ID NO:226) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:226 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:226 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:226 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NO:226 is fully modified with a PS backbone.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:226 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:226 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:226 is represented by SEQ ID NO:227: eGseGseC*seAseAsdTsdUsdC*sdTsdTsdTsdC*sdTsdTsdGseAseAseA s eT (SEQ ID NO:227)
  • the antisense oligonucleotide comprises the following base sequence: TGGCAATUCTTTCTTGAAAA (AON94) (SEQ ID NO: 228)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:228.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:228 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:228 present in the gap of said oligonucleotide (C at position 9 and at position 13 of SEQ ID NO:228) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:228 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:228 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:228 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NO:228 is fully modified with a PS backbone.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:228 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:228 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:228 is represented by SEQ ID NO:229: eTseGseGseC*seAsdAsdTsdUsdC*sdTsdTsdTsdC*sdTsdTseGseAseAseA s eA (SEQ ID NO:229)
  • the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON95) (SEQ ID NO: 230)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:230.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NQ:230 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NQ:230 present in the gap of said oligonucleotide (C at position 7 and at position 1 1 of SEQ ID NQ:230) are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NQ:230 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NQ:230 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate).
  • the oligonucleotide comprising SEQ ID NQ:230 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings.
  • the oligonucleotide comprising SEQ ID NQ:230 is fully modified with a PS backbone.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:230 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:230 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NQ:230 is represented by SEQ ID NO:231 : eGseC*oeAoeAoeTsdUsdC*sdTsdTsdTsdC*sdTsdTsdGsdAseAoeAoeA o eTseG (SEQ ID NO:231)
  • the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON96) (SEQ ID NO: 232)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:232.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
  • At least one C of the oligonucleotide comprising SEQ ID NO:232 is 5- methylcytosine.
  • the C of the oligonucleotide comprising SEQ ID NO:232 present in the gap of said oligonucleotide are not methylated.
  • all Cs of the oligonucleotide comprising SEQ ID NO:232 are 5-methylcytosines.
  • the oligonucleotide comprising SEQ ID NO:232 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:232 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NO:232 is fully modified with a PS backbone.
  • At least one internucleoside linkages in one wing is not modified and is thus a phosphodiester internucleoside linkage.
  • a preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
  • one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:232 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide.
  • one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:232 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
  • a most preferred oligonucleotide comprising SEQ ID NO:232 is represented by SEQ ID NO:233: eGseGoeC*oeAoeAsdTsdUsdC*sdTsdTsdTsdC*sdTsdTsdGseAoeAoeA o eAseT (SEQ ID NO:233)
  • the antisense oligonucleotide comprises the following base sequence: TGGCAATUCTTTCTTGAAAA (AON97) (SEQ ID NO: 234)
  • the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:234.
  • the length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.

Abstract

The invention relates to the field of human genetics, more specifically to treatments for a disease or condition associated with an abnormal processing of the Amyloid Precursor Protein (APP), preferably familiar Alzheimer disease (FAD). The invention in particular relates to antisense oligonucleotides (AONs) that can be used for treating such diseases or conditions.

Description

Antisense oligonucleotides for treating a disease or condition associated with an abnormal processing of APP
Field
The invention relates to the field of human genetics, more specifically to treatments for a disease or condition associated with an abnormal (or altered) processing of the Amyloid Precursor Protein (APP), preferably familiar Alzheimer disease (FAD). The invention in particular relates to antisense oligonucleotides (AONs) that can be used for treating such diseases or conditions.
Background of the invention
Neurocognitive disorders include a broad category of degenerative brain diseases marked by a progressive decline in memory, learning, perception, social, recognition, orientation, language, comprehension, and judgment (American Psychiatric Association, 2013, 5th. Ed. Arlington). Neurocognitive disorders are a manifestation of impaired multiple molecular pathways and cellular functions, leading to synapse loss, cell death, inflammation, gliosis, and disruption of the functional networks that underlie cognition, sensation, and motor skills (Elahi & Miller, 2017 Nature Reviews. Neurology, 13:457-476). The continuous decline in physical function in patients ultimately leads to loss of autonomy and death. Alzheimer's disease (AD) is the most common neurocognitive and neurodegenerative disease, and the most frequent cause of dementia (60% to 80% of cases). AD involves both monogenic and complex forms of inheritance, and is characterized by the accumulation of insoluble amyloid-p plaques (also called Ap plaques) in extracellular spaces and vascular walls, the aggregation of the microtubule protein Tau in neurofibrillary tangles in neurons, dystrophic neurites, loss of synapses, neurotransmitter alterations (acetylcholine deficiency and glutamate excitotoxicity), and micro- and astrogliosis. Molecular and cellular pathological events start decades before dementia becomes apparent. The age of onset of AD is over 65 years of age with risk polymorphisms in genes that regulate microglial immune activation, lipid metabolism and endocytosis. (Masters et al., 2015 Nature Reviews, 1 : 1-18; Congdon et al 2018, Nature Reviews Neurology, 14(7), 300-415); Long and Holtzman et al., 2019 Cell, 170(2), 312-339).
Familial Alzheimer’s disease (FAD) is an Early-Onset AD (EOAD) (before 65 years of age) caused by mutations in genes involved in A peptide production: APP (OMIM 104760), PSEN1 (OMIM 104311), or PSEN2 (OMIM 1600759) (Lanoiselee et al., 2017, PLoS Medicine, 14:3). FAD has an autosomal dominant inheritance pattern and a prevalence of 1 % of all AD cases. In FAD patients Ap plaques depositions are detected in asymptomatic patients and most likely drive the progression of Tau pathology. High rates of Tau accumulation can be seen in symptomatic FAD patients and Tau pathology has been suggested as an indicator of the onset of cognitive impairments (Gordon et al., Brain. 2019 Apr 1 ;142(4):1063-1076). In humans, it has been suggested that Ap plaques deposition does not correlate with the degree of cognitive decline but may be required for progression of Tau pathology.
Ap is produced by sequential cleavage of p-amyloid precursor protein (APP) by p-secretase and y-secretase. The p-secretase enzyme (BACE1) cleaves APP at the N terminus of the Ap sequence, releasing secreted APP-p and the membrane-bound C99 fragment. Following cleavage by BACE1 , the y-secretase complex binds to N-terminally cleaved APP fragment (C99) and intramembranous cleaves at the s-site releasing C-terminal fragment (CTF) and Ap49 or Ap48. The y-secretase complex then processes along the remaining Ap C-terminal end, producing sequentially shorter peptides until the Ap peptide is released from the complex (generally after producing peptides 38-, 40-, and 42-amino acids in length). Therefore, the processivity of the y-secretase complex defines the length of the generated Ap peptide. Ap peptides are prone to aggregate into p sheet conformations in the form of higher-order oligomers, protofibrils, and fibrils, which are detectable in AD brain. Longer Ap peptides (> Ap42) are more hydrophobic and show more capacity to aggregate and form Ap plaques (L. Chavez-Gutierrez and M. Szaruga, 2020, Seminars in Cell and Developmental Biology 105, 75-85; Long and Holtzman et al., 2019, Cell 170, 312-339) Importantly, y-secretase complexes exhibit broad substrate specificity with close to 100 type 1 membrane proteins listed as potential substrates, besides APP (Wakabayashi et al., 2008, Physiology 23, 194-204).
The y-secretase complex consists of four protein subunits: Presenilin (PSEN), presenilin enhancer 2 (PEN-2), Anterior pharynx-defective (APH), and Nicastrin. There are multiple isoforms of PSEN (PSEN1 or PSEN2) and APH (APH1A or APH1 B); therefore up to four different y- secretase complexes may exist in a single cell (De Strooper., 2003 Neuron, 38, 9-12). PSEN1 is an aspartyl protease and is the catalytic subunit of y-secretase, PEN-2 is required for y-secretase maturation, APH stabilizes the complex and Nicastrin is thought to play a role in substrate binding (De Strooper et al., 1998, Nature 391 , 387-390, De Strooper et al., 1999, Nature 398, 518-522, Wolfe at al., 1999, Nature 398, 513-517; N. Takasugi et al., 2003, Nature 422, 438-441 ; S. Shah et al., 2005, Cell 122, 435-447; R. Zhou et al., 2019, Science 363, 6428).
The proteins involved in Ap peptide production form part of a membrane-embedded protease complex called the y-secretase complex and are composed of the following four proteins with a 1 :1 :1 :1 stoichiometry: Nicastrin (NCT), Anterior pharynx-defective 1 (APH1A or APH1 B), Presenilin enhancer 2 (PEN-2) and Presenilin (PSEN-1 or PSEN-2). Presenilin-1 and -2 are the catalytic subunits of the complex (De Strooper., 2003Neuron, 38, 9-12). Presenilin 1 and Presenilin 2 are paralogs, y-secretase complexes exhibit broad substrate specificity with close to 100 type 1 membrane proteins listed as potential substrates (Wakabayashi et al., 2008, Physiology 23, 194-204). One of the substrates of the y-secretase complex is the Amyloid Precursor Protein (APP), which is converted into Ap peptides by said complex (De Strooper et al., 1998, Nature 391 , 387-390). APP may be processed through two distinct pathways: the non- amyloidogenic pathway and the amyloidogenic pathway.
Mutations in APP (OMIM 104760), PSEN1 (OMIM 104311) and PSEN2 (OMIM 1600759), have been found to be the main cause of the Familial Alzheimer’s disease (FAD) (Lanoiselee et al., 2017, PLoS Medicine, 14:3). Currently, almost 300 mutations in PSEN1 , over 10 in PSEN2 and over 18 in APP, have been linked to early onset FAD. Mutations in PSEN1 are not only significantly more prevalent than the ones in PSEN2 or APP, but are also associated with earlier clinical onsets and atypical cognitive presentations, such as motor impairments (L. Chavez- Gutierrez and M. Szaruga, 2020, Seminars in Cell and Developmental Biology 105, 75-85); Mutations in PSEN1 have different effects:
1 . some reduce or abolish y-secretase activity,
2. others decrease the processivity of the y-secretase activity, thereby shifting the y- secretase activity towards generation of longer Ap peptides species from APP,
3. others reduce the stability of the y-secretase and the A peptide during the sequential cleavage, or
4. reduce PSEN1 maturation.
It is strongly debated whether presenilin mutations cause Alzheimer’s Disease by loss of function or gain of function (Xia et al., 2015, Neuron, 85(5), 967-81 ; Veugelen et al., 2016, Neuron, 90(2), 410-6). The most parsimonious explanation is that mutations in PSEN1 affect the activity of y- secretase, leading to an increase in Ap42/40 ratio, which increases neurotoxic oligomer assembly that causes synaptic dysfunction and neurodegeneration (Zoltowska & Berezovska, 2017, Molecular Neurobiology 55, 2275-2284, Chavez-Gutierrez et al., 2012. The EMBO Journal 31 , 2261-2274; Szaruga et al., 2017, Cell 170, 443-456).
Until 2021 , only symptomatic treatments were available for AD patients, mainly comprising cholinesterase inhibitors and NMDA inhibitors (Long and Holtzman et al., 2019, Cell 170(2), 312- 339). On June 7th 2021 , the U.S. Food and Drug Administration (FDA) granted accelerated approval of the first disease-modifying treatment for AD: Aduhelm (aducanumab). However, a post-approval trial is required to verify that the drug provides the expected clinical benefit and grant a continued approval for Aduhelm. Adulhelm is a monoclonal antibody that targets extracellular Ap plaques in the brain. (https://www.fda.gov/news-events/press- announcements/fda-grants-accelerated-approval-alzheimers-drug)
Therefore, there is still a need for new and more efficient treatments for a disease or condition associated with an abnormal (or altered) processing of the Amyloid Precursor Protein (APP), preferably familiar Alzheimer disease (FAD). The inventors designed antisense oligonucleotides that could be used for such treatment. Such antisense oligonucleotides are expected not to have all drawbacks of existing treatments.
Legends to the figures
Figure 1.
Analysis showing the effect on PSEN1 protein levels (A) and gamma-secretase activity (B-D) of AON20 (SEQ ID NO:1 18) treatment to Tg/Tg A431 E mouse neurons. (A -B) Western blot analysis showing the protein levels of full-length PSEN1 (A) or the protein levels of CTF APP normalized by full-length APP (B) after 8 days treatment with AON20 at the indicated concentrations. Data in A and B is shown relative to the non-treated condition (OpM). (C-D) ELISA analysis showing the profile of soluble Ap38, Ap40 and Ap42 species in the supernatant of mouse Tg/Tg A431 E neurons after 8 days treatment with AON20 at the indicated concentrations. Data is shown as Ap levels (C) and as Ap42/38 ratio (D). Each dot in the graph corresponds to one cell culture well, error bars indicate standard deviation (n=2-3). * p < 0.05, ** p <0.01 (one-way ANOVA test). CTF= C-terminal fragment. FL = full length.
Figure 2.
ELISA analysis showing the profile of soluble Ap38, Ap40, and Ap42 species in the supernatant of mouse Tg/Wt A431 E neurons after 7d treatment with AON21 (SEQ ID NO:120) at the indicated concentrations. Data is shown as Ap levels (A) and as Ap42/38 ratio (B). Each dot in the graph corresponds to one cell culture well, error bars indicate standard deviation (n=3). * p < 0.05, ** p <0.01 (one-way ANOVA test).
Figure 3.
Wes analysis showing the effectivity and allele specificity of AONs targeting human A431 E PSEN1 transfected to NIH3T3 cells overexpressing Flag-tagged human WT or A431 E PSEN1. Data is shown as the ratio of Flag and NPT-II protein levels normalized to non-treated samples. AONs were transfected at 10nM and cells were harvested 24h aftertransfection. (A) Protein levels of WT and A431 E PSEN1 after treatment with AONs wherein the sequence and the position of the nucleotide opposite the mutation shifted from the 5’ end to the 3’ end. (B) Protein levels of WT and A431 E PSEN1 after treatment with derivatives from AON4, AON5, and AON6 (SEQ ID NO:8, 10 and 12) which contain a T (thymine) to U (uracil) substitution at the position opposite the mutation. (C) Backbone modifications used in AONs listed in Table 4. (D) Protein levels of WT and A431 E PSEN1 after treatment with derivatives from AON14 (SEQ ID NO:28) that incorporate PO and/or PNms linkages. NT = non-treated. Each dot in the graph corresponds to one cell culture well, error bars indicate standard deviation. Figure 4.
Analysis showing the effect of the position of the mutation within the AON and the effect of a T (thymine) to U (uracil) substitution opposite to the mutation position on the effectivity and allele selectivity of AONs targeting human A431 E (A) or S212Y mutations (B). NIH3T3 cells were transfected with plasmids encoding for WT, A431 E or S212Y human PSEN1. Three hours later, 10nM of the indicated AONs were transfected and cells were harvested 24h after transfection. (dd)PCR was used to measure WT and mutant mRNA levels. Data is shown as the ratio of WT and A431 E (A) or S212Y (B) PSEN1 mRNA levels. AONs used are listed in Table 5. Each dot in the graph corresponds to one cell culture well (n=1 or 2), error bars indicate standard deviation.
Figure 5.
(dd)PCR analysis showing mRNA levels of WT and A431 E PSEN1 in human neurons differentiated from patient-derived iPSC after 10 days of treatment with AON14 (SEQ ID NO:28), AON15 (SEQ ID NO:30), and AON16 (SEQ ID NO:33). mRNA copies of WT and A431 E were quantified with a SNP assay and normalized to the mRNA copies of two housekeeping genes (TFRC and GAPDH). Data is shown relative to the non-treated condition (NT), n=1 cell culture well.
Figure 6.
Analysis showing the effect on WT and A431 E mouse Psenl mRNA levels (A) and y-secretase activity (B-D) of AON87 (SEQ ID NO: 202) treatment to Tg/Wt A431 E primary mouse neurons. AON87 targets specifically mouse WT Psenl . (A) RT-qPCR analysis showing the mRNA levels of A431 E and WT mouse Psenl after 7 days of treatment with AON87 at the indicated concentrations. mRNA levels of A431 E and WT Psenl were normalized to the levels of two housekeeping genes (Actin and Rsp23). Data is shown relative to the non-treated condition (OpM). (B-D) ELISA analysis showing the profile of soluble Ap38, Ap40, and Ap42 species in the supernatant of Tg/ Wt A431 E primary mouse neurons after 7 days of treatment with AON87 at the indicated concentrations. Data is shown as total Ap levels (B), as levels of Ap38, Ap40, and Ap42 relative to the non-treated condition (C), and as the ratio Ap42/38 (D). mRNA and soluble Ap levels were measured in the same samples, each dot in the graph corresponds to one cell culture well (n=9), error bars indicate standard deviation. **** p <0.0001 (A and C, two-way ANOVA test with Dunnett's multiple comparisons test; B and D, t-test).
Figure 7.
RT-qPCR analysis showing mRNA levels of WT and A431 E Psenl in Tg/Tg (A) and Tg/Wt (B) mouse primary neurons after 7 days of treatment with AQN20 (SEQ ID NO:118) and AON88 (SEQ ID NQ:204) at the indicated concentrations. Data is shown relative to the non-treated condition (OpM). Each dot in the graph corresponds to one cell culture well (n=9), error bars indicate standard deviation. *** p <0.001 , **** p <0.0001 (one-way ANOVA test with Dunnett's multiple comparisons test). Figure 8.
ELISA analysis showing the profile of soluble Ap38, Ap40, and Ap42 species in the supernatant of Tg/Wt primary mouse neurons after 7 days of treatment with AON20 (SEQ ID NO:118) and AON88 (SEQ ID NO: 204) at 10 pM concentration. Data is shown as total Ap levels (A), as levels of Ap38, Ap40, and Ap42 relative to the non-treated condition (B), and as the ratio A042/38 (C). Each dot in the graph corresponds to one cell culture well (n=9), error bars indicate standard deviation. *** p <0.001 , **** p <0.0001 (A and C, one-way ANOVA test with Dunnett's multiple comparisons test; B, two-way ANOVA test with Dunnett's multiple comparisons test). NT: none treated.
Figure 9.
Analysis showing AON20 (SEQ ID NO:118) efficacy and allele selectivity in-vivo. Psenl wt/A43i E; pp i mjce recejve a single intracerebroventricular injection of 150 pg of AQN20. Animals were sacrificed after 2 weeks and hippocampal mRNA levels of WT and A431 E Psenl were measured by RT-qPCR. Results were normalised to Actin and GAPDH levels. Data is shown relative to vehicle condition. Each dot in the graph corresponds to one animal (n=7, vehicle; n=4, AQN20), error bars indicate standard deviation. Statistical test: T-test.
Description of the invention
The invention targets the real underlying source of FAD by depleting the mutant form of the allele and in that way restoring the normal activity of the catalytic subunit of the y-secretase complex using an oligonucleotide of the invention as defined herein. Such oligonucleotide preferentially targets and breaks down the allele carrying a mutation in PSEN1 or PSEN2, which will reduce the levels of mutant PSEN1 or PSEN2, allow replacement by the corresponding wild type proteins, increase the levels of functional y -secretase, and thus reduce the production of toxic forms of Ap peptides. This strategy is different from attempts to inhibit or downregulate the y-secretase as such which causes severe (Notch-)signaling side effects and is also expected to be more effective than current approaches that focus on more downstream features of the disease such as Tau aggregation or more generic features of the disease like inflammation for the treatment of familial AD. This strategy is quite unique: the oligonucleotides are selected to have the highest possible efficiency and the highest possible specificity against the mutated allele of PSEN1 or PSEN2, while they are selected for the lowest possible efficiency and the lowest possible specificity against the wild type allele of PSEN1 or PSEN2. It means the oligonucleotides have been designed to specifically inactivate, delete, knock down, suppress the mutated allele of the PSEN1 or PSEN2 and primarily keep the wild type allele thereof intact.
Antisense oligonucleotide
The inventors surprisingly discovered antisense oligonucleotides that exhibit attractive therapeutic activities and that may be used for treating a disease or condition associated with an abnormal (or altered) processing of the Amyloid Precursor Protein (APP) preferably familiar Alzheimer disease (FAD).
Such antisense oligonucleotides are described below in more detail. Such antisense oligonucleotides will be referred to herein as antisense oligonucleotides according to the invention (or antisense oligonucleotide of the invention or oligonucleotide of the invention or oligonucleotide or AON).
In a first aspect, there is provided an antisense oligonucleotide which preferentially targets a mutated allele of a protein of the y-secretase complex when present in a cell comprising (preferably expressing) said mutated allele. In a preferred embodiment, the protein of the y- secretase complex whose allele is mutated is PSEN1 or PSEN2, preferably PSEN1 . In a preferred embodiment, the antisense oligonucleotide is single-stranded.
In a second aspect, there is provided an antisense oligonucleotide comprising 15 to 30 or 20 to 30 nucleotides including a central region of 5 to 15 consecutive DNA nucleotides and/or DNA nucleotides analogues flanked in each end by wing regions comprising 1 to 5 RNA nucleotides analogues, which preferentially targets a mutated allele of a protein of the y-secretase complex when present in a cell comprising and preferably expressing said mutated allele, wherein the mutation present in the mutated allele is targeted by the first, second, third, fourth, fifth, sixth, seventh, eight, nineth, tenth, eleventh, or twelfth nucleotide of the central region of the oligonucleotide. In a preferred embodiment, the protein of the y-secretase complex whose allele is mutated is PSEN1 or PSEN2, preferably PSEN1. In a preferred embodiment, the antisense oligonucleotide is single-stranded.
As demonstrated in the example section of the present application, the antisense oligonucleotides of the second aspect are demonstrated to efficiently target a mutated allele of a protein of the y- secretase complex, such as PSEN1 .
In an embodiment, there is provided an antisense oligonucleotide comprising 15 to 30 or 20 to 30 nucleotides including a central region of 5 to 15 consecutive DNA nucleotides and/or DNA nucleotides analogues flanked in each end by wing regions comprising 1 to 5 RNA nucleotides analogues, which preferentially targets a mutated allele of a protein of the y-secretase complex when present in a cell comprising and preferably expressing said mutated allele, wherein the mutation present in the mutated allele is targeted by the sixth, seventh, eighth, nineth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, or eighteenth nucleotide in the oligonucleotide sequence starting from 5’ to 3’. In a preferred embodiment, the protein of the y-secretase complex whose allele is mutated is PSEN1 or PSEN2, preferably PSEN1. In a preferred embodiment, the antisense oligonucleotide is single-stranded.
All definitions and/or embodiments described herein relate to both the first and the second aspects of the invention unless otherwise indicated.
In an embodiment, said cell is a cell, wherein said protein is endogenously expressed (mutated allele and preferably the wild type allele). In a preferred embodiment, the cell is a mammalian cell, preferably a murine or human cell. In a more preferred embodiment, the cell is representative for the disease FAD.
In a first embodiment, a cell which is representative for the disease FAD is a neuronal cell. Preferred neuronal cells are cells of the central nervous system. A preferred cell of the central nervous system may be from, or may be derived from, a region of the brain which is known to be affected in FAD. Such brain regions include the hippocampus, amygdala, cerebral cortex (e.g frontal lobe, parietal lobe, temporal lobe and/or occipital lobe) cerebellum, and the thalamus. Accordingly, in an embodiment, the activity of the antisense oligonucleotide is assessed in such neuronal cells. The assessment may also be carried out in vitro by culturing such cells and contacting them with the oligonucleotide.
In a second embodiment, a cell which is representative for the disease FAD is a human iPSC (induced pluripotent stem cell), which is triggered to differentiate or mature into neurons. As indicated in Example 5, the antisense oligonucleotides of the invention are able to successfully target proteins of the y-secretase complex whose allele is mutated (PSEN1) in biologically relevant FAD systems, such as iPSC cells. Preferably, the iPSC is from a subject having an altered or diminished PSEN1 or PSEN2 activity, and is a FAD patient or is suspected to become a FAD patient due to his/her genetic background. Accordingly, in an embodiment, the activity of the antisense oligonucleotide is assessed using such neuronal cells derived from human iPSC. The assessment may be carried out in vitro by culturing such cells and contacting them with the oligonucleotide.
Alternatively in another embodiment, said cell is a cell, wherein said protein is exogenously expressed (mutated allele or the wild type allele). In a preferred embodiment, the cell is a mammalian cell, preferably a murine or human cell. In this embodiment, a nucleic acid coding for said mutated protein is introduced into said cell. Said cell may be a non-neuronal cell, including but not limited to fibroblast, HeLa, or HEK293 cell. Said nucleic acid coding for said mutated protein may be introduced by viral or non-viral vectors, more preferably plasmids expressing the mutant protein or fusion-derivatives thereof.
Depending on the oligonucleotide used, the skilled person will know which cell is suitable to assess the activity of the oligonucleotide. As illustrated above, in an embodiment, said cell is a cell wherein said protein is endogenously expressed. Alternatively in another embodiment, a nucleic acid coding for said mutated protein is introduced into said cell. The skilled person may also assess the activity of the oligonucleotide in a cell exogenously and subsequently in a cell endogenously expressing the mutated protein.
The choice of the read out is a key feature of the invention as the use of a given read out may lead to the identification of oligonucleotide that are not optimal when later validated using a more relevant read out. Here, the inventors demonstrated that the use of the effect of the oligonucleotide on the mutated PSEN1 or PSEN2 transcript and/or the absence of effect on the wild type allele of said transcript and/or the effect of the oligonucleotide to normalize, reverse or correct abnormal A042/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios, preferably the Ap42/38 and/or Ap43/40 ratios were crucial, more preferably Ap42/38 ratios were crucial. In particular, the inventors have surprisingly found that antisense oligonucleotides of the invention have the biggest impact on Ap38 peptide levels and the Ap42/Ap38 ratio while the effect on other Ap-species (such as Ap40) is less pronounced (Examples 2, 6, and 8). This observation confirms the need of using holistic readouts to evaluate the therapeutic benefit of the therapeutic effect of antisense oligonucleotides;
In a preferred embodiment, there is provided an antisense oligonucleotide which preferentially targets a mutated allele of a protein of the y- secretase complex (preferably PSEN1) when present in a cell comprising (preferably expressing) said mutated allele. More preferably, the cell is a neuronal cell, even more preferably a human neuronal cell. Preferably, the cell expresses the endogenous mutated allele of a protein of the y-secretase pathway. In a preferred embodiment, - the oligonucleotide is able to preferentially silence, inactivate, break down, knock down, reduce or decrease mutated PSEN1 or PSEN2 transcripts (preferably mutated PSEN1 transcripts) and even more preferably
- the oligonucleotide is not (or minimally) able to silence, inactivate, break down, knock down, reduce or decrease wild-type PSEN1 or PSEN2 transcripts (preferably wild-type PSEN1 transcripts).
In a more preferred embodiment,
- the oligonucleotide is able to preferentially silence, inactivate, break down, knock down, reduce or decrease mutated PSEN1 or PSEN2 transcripts (preferably mutated PSEN1 transcripts), and even more preferably the oligonucleotide is not (or minimally) able to silence, inactivate, break down, knock down, reduce or decrease wild-type PSEN1 or PSEN2 transcripts (preferably mutated PSEN1 transcripts) and/or
- the oligonucleotide is able to normalize, reverse or correct abnormal Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios, preferably the Ap42/38 and/or Ap43/40 ratios, and more preferably the Ap42/38 ratio.
In an embodiment, the oligonucleotide:
1 . preferentially silences or inactivates or breaks down mutated PSEN1 transcripts, and knocks down or reduces or decreases mutant PSEN1 protein expression and preferentially does not silence or inactivate or break down wild-type PSEN1 transcripts, and does not knock down or reduce or decrease wild-type PSEN1 protein expression, and/or
2. is able to normalize, correct, or reverse abnormal Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably Ap42/38 and/or Ap43/40 ratios, and more preferably the Ap42/38 ratio.
In an embodiment, the oligonucleotide:
1 . preferentially silences or inactivates or breaks down mutated PSEN1 transcripts, and/or preferentially does not silence or inactivate or break down wild-type PSEN1 transcripts, and/or
2. is able to normalize, correct, or reverse abnormal Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably Ap42/38 and/or Ap43/40 ratios, more preferably the Ap42/38 ratio).
Each of these read outs have been extensively defined below.
In the context of the invention, an activity elicited or exhibited by an antisense oligonucleotide of the invention is to preferentially target a mutated allele of a protein of the y-secretase complex when present in a cell comprising said mutant allele. In an embodiment, said protein is a human protein and/or is PSEN1 .
The targeted mutated allele, especially the region or stretch of this mutated allele which is targeted may also be named the targeted region of the oligonucleotide.
This mutated allele of a human protein of the y- secretase complex may be a disease-associated or disease-causing allele in a cell of a patient, in a tissue of a patient and/or in a patient as explained later herein. In an embodiment, the patient is a mammal. In a preferred embodiment, the patient is a human.
Throughout the application, the word “binds”, “specifically binds”, “targets”, “specifically targets”, “hybridizes” or “specifically hybridizes” could be used interchangeably when used in the context of an antisense oligonucleotide which is reverse complementary to a part of an allele, especially in the form of a transcript encoding this allele as identified herein. It follows that the expression “oligonucleotide” is synonymous with the expression “antisense oligonucleotide” in the context of the application.
Throughout the application, the word “preferentially binds”, “preferentially targets” or “preferentially hybridizes” could be used interchangeably when used in the context of an antisense oligonucleotide of the invention which is reverse complementary to a part of a mutated allele, especially in the form of a transcript encoding this mutated allele as identified herein. This “preferential binding/targeting/hybridizing” is later herein defined by comparison to the “specific binding/targeting/hybridizing” to a wild type/control/non mutated allele.
The word “transcript” refers to the pre-mRNA or mRNA encoded by an allele. In the context of the invention, a mutated allele is a gene encoding a mutated transcript, said mutated transcript encoding a mutated protein of the y- secretase complex.
In this context, the wild type genomic DNA of mouse PSEN1 comprises SEQ ID NO: 145, the corresponding coding RNA comprises SEQ ID NO:146 and the mouse PSEN1 protein comprises SEQ ID NO:147.
In this context, the A431 E genomic DNA of mouse A431 E PSEN1 comprises SEQ ID NO: 148, the corresponding coding RNA comprises SEQ ID NO:149 and the mouse A431 E PSEN1 protein comprises SEQ ID NO:150.
In this context, the wild type genomic DNA of human PSEN1 comprises SEQ ID NO: 151 , the corresponding coding RNA comprises SEQ ID NO:152 or 169 and the human PSEN1 protein comprises SEQ ID NO:153 or 159.
In this context, the A431 E genomic DNA of human A431 E PSEN1 comprises SEQ ID NO: 154, the corresponding coding RNA comprises SEQ ID NO:155 or 170 and the human A431 E PSEN1 protein comprises SEQ ID NO:156 or 161.
In this context, the wild type genomic DNA of human PSEN2 comprises SEQ ID NO: 165, the corresponding coding RNA comprises SEQ ID NO: 171 or 172 and the human PSEN2 protein comprises SEQ ID NO:166 or 168. As used herein, "hybridization" refers to the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid/target region). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases). For example, the natural base adenine is nucleobase complementary to the natural nucleobases thymine, 5-methyluracil and uracil which pair through the formation of hydrogen bonds. The natural base guanine is nucleobase complementary to the natural bases cytosine and 5-methyl-cytosine. Hybridization can occur under varying circumstances. In particular, hybridization of an oligonucleotide of the invention with a targeted pre-mRNA and/or mRNA can occur under varying circumstances. Similarly, binding of an oligonucleotide of the invention to a targeted pre-mRNA and/or mRNA (i.e. targeted region) can occur under varying circumstances. Preferably, said hybridization or said binding is assessed under physiological conditions in a cell, more preferably in a human cell. Preferred cells have been defined earlier herein. An oligonucleotide of the invention is preferably said to be able to bind to, or capable of binding to, or able to hybridize with, or capable of hybridizing with, when said binding or hybridization occurs under physiological conditions in a cell, preferably a human cell. Preferred cells have been defined earlier herein.
In the context of the invention, “hybridizes” or “binds” (or “preferentially hybridizes” or preferentially binds” or “preferentially binds” or “preferentially hybridizes”) is used under physiological conditions in a cell, preferably a human cell unless otherwise indicated.
In an embodiment, an antisense oligonucleotide is said to target a mutated allele encoding a mutated protein of the y-secretase complex (preferably PSEN1) when said oligonucleotide is reverse complementary to at least 10 or at least 15 consecutive/contiguous bases of said mutated allele or of said mutated transcript encoding said mutated protein. This “at least 10 or at least 15 consecutive/contiguous bases of said mutated allele” may also be called the targeted region or the target region of the antisense oligonucleotide. In an embodiment, the length of the reverse complementary part may be at least 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 nucleotides. Within the context of the invention, an oligonucleotide comprises up to 50 nucleotides and/or nucleotide analogues. An oligonucleotide may be 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides and/or nucleotide analogues. The part of the oligonucleotide which is reverse complementary does not per se need to be 100% reverse complementary with the mutated allele or transcript of a mutated protein of the y-secretase complex. In an embodiment, one ortwo mismatches may be present over a length of at least 15 nucleotides in total defining the part which is reverse complementary with said mutated allele or transcript.
An activity of the oligonucleotide of the invention is to preferentially target a mutated allele or mutated transcript encoding a mutated protein of the y-secretase complex. This activity may mean that (the levels of) the targeted mutant allele (or corresponding transcript) is silenced or knocked down (or broken down) or inactivated or reduced or decreased. In other words, the levels of said mutated transcript are knocked down (or broken down), reduced or decreased. In this context, the protein of the y-secretase complex is PSEN1. In a preferred embodiment, the protein is a human protein. More preferably, human PSEN1.
Therefore, a first way of assessing an activity of the antisense oligonucleotide of the invention is to assess whether the oligonucleotide is able to knock down (or break down) or reduce or decrease (the levels of) said mutated transcript (preferably PSEN1 or PSEN2 mutated allele or transcript). This reduction or decrease of the level of mutated transcript may be of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the level of the same transcript at the onset of the treatment. In a preferred embodiment, the mutated transcript is no longer detectable. The reduction or decrease may be assessed in cells or tissues or subjects treated with said oligonucleotide and using Northern Blotting or (semi) quantitative RT-qPCR or RT-ddPCR analysis of mutant transcript copy numbers (preferably as carried out in the experimental part).
In a preferred embodiment, for this first way of assessing an activity of the antisense oligonucleotide of the invention, the level of the wild type allele or wild type transcript which is not targeted (or not specifically targeted) by the oligonucleotide is also assessed. Accordingly, the wild type allele or transcript is still detectable and the level of said wild type allele or transcript is not less than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the level of the same allele or transcript at the onset of the treatment. In a preferred embodiment, the level of the wild type allele or transcript is the same as the one detected at the onset of the treatment. The presence of said transcript may be assessed in cells or tissues or subjects treated with said oligonucleotide and using Northern Blotting or (semi) quantitative RT-qPCR or RT-ddPCR analysis of mutant transcript copy numbers (preferably as carried out in the experimental part).
Therefore, in a preferred embodiment, the activity of the antisense oligonucleotide of the invention is assessed as follows:
- whether the oligonucleotide is able to knock down or reduce or decrease the levels of a mutated PSEN1 or PSEN2 transcript and
- whether the oligonucleotide is not (or minimally) able to knock down or reduce or decrease the levels of a wild type PSEN1 or PSEN2 transcript.
This reduction or decrease of the level of mutated transcript may be of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the level of the same transcript at the onset of the treatment. In a preferred embodiment, the mutated transcript is no longer detectable.
The oligonucleotide is said not (or minimally) be able to knock down or reduce or decrease the wild type PSEN1 or PSEN2 allele or transcript when said wild type allele or transcript is still detectable is not less than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the level of the same allele or transcript at the onset of the treatment. In a more preferred embodiment, the oligonucleotide is able to knock down or reduce or decrease the levels of a mutated PSEN1 or PSEN2 transcript of at least 20 or at least 30 or at least 40 or at least 50 or at least 80 or at least 90 or at least 95% compared to the level of said mutated allele or transcript at the onset of the treatment and the levels of a wild type PSEN1 or PSEN2 transcript is still at least 50 or at least 80 or at least 90 or at least 95% of the level of said transcript of allele at the onset of the treatment.
The presence of a mutated or of a wild type allele or transcript is assessed as earlier described herein.
As explained in the background section, some mutations in PSEN1 or PSEN2 gene may shift y- secretase complex activity towards the production of abnormal Ap profiles, from short peptides (A 38, A 40) to longer peptides (A 42, A 43 and even longer up to A 48 or A 49, although there are no tools readily available to measure those species while the other ones are readily measurable with ELISA assays), as the result of an impairment of the carboxypeptidase-like activity of y-secretase. The underlying mechanism is the destabilization of the substrate-enzyme complex i.e. the APP substrate interaction with the catalytic presenilin subunit in the y-secretase complex is decreased because of the mutation of the PSEN1 or PSEN2 gene. This results in faster release of longer (less processed) forms of A i.e. any peptide with a length up to 49 amino acids. The longer (larger or equal to Ap49 or larger or equal to Ap48 larger or equal to Ap42 or larger or equal to Ap43) forms of the Ap peptide species are toxic because they are more hydrophobic and aggregate more easily and form more easily toxic oligomeric and fibrillic species. The processing of Ap is progressive and there are two production lines, one starting with Ap49>46>43>40>37 and one starting at Ap48>45>42>38 (Takami et al., 2009, J Neurosci 29, 13042-52). In other words, Ap42/38 and/or Ap 43/40 and/or Ap43/37 and/or Ap 42/40 ratios are expected to increase in brain, cerebrospinal fluid (CSF) and/or plasma of FAD patients because of the PSEN1 or PSEN2 mutations. In an embodiment the Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio) in the plasma, and/or CSF and/or brain of FAD patients is increased of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to the level in healthy subjects. The Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio) may be assessed by techniques known to the skilled person.
In an embodiment, the Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio) may be assessed using cell extract, cell medium, brain extract, CSF or plasma of FAD patients carrying PSEN1 or PSEN2 mutations.
In an embodiment, the Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio) may be assessed using cell extract, cell medium of cells endogenously or exogenously expressing a mutated PSEN1 or PSEN2.
In an embodiment it is assessed using ELISA, preferably as carried out in the experimental part.
In the in vivo situation (in transgenic mouse), these ratios in CSF, plasma and/or in brain extracts are first expected to increase and subsequently to decrease as a consequence of the aggregation of the Ap peptides forming Ap plaques.
In the in vivo situation (in FAD patients), these ratios in in CSF and/or plasma are first expected to increase and subsequently to decrease as a consequence of the aggregation of the A peptides forming Ap plaques.
This increase is typically observed in FAD patients with no detectable Ap plaques in the brain. FAD patients with no detectable Ap plaques in the brain may be called asymptomatic FAD patients. During the progression of the disease, Ap plaques are formed and the ratios decreased in CSF samples . In plasma, these ratios are increased during all the progression of the disease. Therefore if the patient already has detectable Ap plaques in the brain, the assay should be carried out in the plasma of said patients. The ELISA assay only assesses the soluble Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio) and the effect of the aggregation process on the level of the ratios cannot be specified. Said ELISA assay primarily visualizes the first effect of the mutation of the y-secretase complex activity, which is the generation of longer forms of the A peptide species.
In an embodiment, an asymptomatic FAD subject or patient is selected or identified in view of his/her family history with several FAD patients having a PSEN1 or PSEN2 mutation. It is crucial to diagnose this type of subject when they are still asymptomatic (and therefore as early as possible) in order to start treatment as early as possible. The skilled person may use alternative in vitro assays that are able to assess the presence of aggregated A peptides in brain extracts from a mouse.
Therefore, a second way of assessing an activity of the oligonucleotide of the invention is to determine the Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, and more preferably the Ap42/38 ratio) in cell cultures, tissues, plasma and/or CSF of treated FAD subjects. In an embodiment, the FAD patients are asymptomatic, with no detectable Ap plaques in the brain. The oligonucleotide is expected to reduce the generation of long Ap species leading to reduction of soluble Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio). When the assessment is carried out in subjects, it is preferably carried out in the plasma and/or CSF of said subjects.
The normalization, correction, or reversion of abnormal Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the A042/38 ratio) may mean a decrease of the A042/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio) of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to its level at the onset of the treatment. The Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio) may be assessed by techniques known to the skilled person. In an embodiment it is assessed using ELISA, preferably as carried out in the experimental part.
If the assessment is carried out in FAD patients with detectable Ap plaques in the brain, it is preferably carried out in the plasma and not in the CSF of said patients.
The invention is uniquely using a read out which directly demonstrates the therapeutic effect of an oligonucleotide on a disease or condition associated with an abnormal (or altered) processing of the APP. This read out directly demonstrates the downstream consequences of the defects of the y-secretase complex present in a cell, in a tissue, in plasma and/or in CSF of a FAD patient by assessing Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio). In an embodiment, the FAD patient is preferably asymptomatic with no detectable Ap plaques in the brain, as earlier defined herein. The effect of the oligonucleotide on this read out is expected to be representative of a therapeutic effect on a patient with a mutated y-secretase complex (preferably PSEN1) that will develop FAD. It is expected that the used read out (i.e. Ap42/38 and/or A 43/40 and/or A 43/37 and/or A 42/40 ratios (preferably the Ap42/38 and/or A 42/40 ratios, more preferably the Ap42/38 ratio)) is representative of the FAD disease and the normalization, correction or reversion of the abnormal increased soluble Ap42/38 and/or A 43/40 and/or Ap43/37 and/or A 42/40 ratios (preferably the Ap42/38 and/or A 42/40 ratios, more preferably the Ap42/38 ratio) is therefore predictive of a therapeutic activity of an oligonucleotide. This read out (any of the soluble Ap42/38 and/or A 43/40 and/or A 43/37 and/or A 42/40 ratios (preferably the Ap42/38 and/or A 42/40 ratios, more preferably the Ap42/38 ratio), also called “a long/short” A ratios) is more representative and more predictive than a read out limited to a single “long” A peptide only (such as soluble Ap42). The goal of the therapy is to restore a normal enzymatic activity of the y- secretase complex or to switch the activity of said complex to a more normal activity. Therefore, the skilled person understands that long/short Ap peptide ratios provide a better representation of the enzymatic activity of the y-secretase complex than the use of just a single long form of the Ap. In other words, in order to better understand, visualize or monitor the activity status of the y- secretase complex (preferably PSEN1), it is preferred to assess the production of several species of the Ap peptides and not only the production of a long form thereof.
In an embodiment, it follows that an antisense oligonucleotide of the invention may be considered to be active when: The oligonucleotide is able to normalize, correct, or reverse abnormal A042/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio), wherein the assessment is compared to the level of the same Ap ratio at the onset of the treatment (or to corresponding level of the same Ap ratios in healthy subjects). The assessment may be carried out in a cell, a tissue, plasma, or CSF or subject. When a subject is treated, said ratios are preferably assessed in plasma of said subjects. A subject may be an asymptomatic FAD subject. Alternatively, the subject may be a symptomatic FAD subject. If the subject is a symptomatic FAD subject, the assessment is preferably carried out using plasma. An alternative way of assessing the effect of the oligonucleotide on the Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio) is applying imaging technologies to assess Ap plaque accumulation in the brain of treated subjects. Imaging technology may include PET scans on FAD patients. Imaging technology may also include histology on murine tissues.
In a preferred embodiment, an antisense oligonucleotide of the invention may be considered to be active when:
- the oligonucleotide is able to preferentially silence, inactivate, breakdown, knock down or reduce or decrease or correct (the quantity or level of) mutated PSEN1 (or PSEN2) transcripts
- the oligonucleotide is not (or minimally) able to silence, inactivate, breakdown, knock down or reduce or decrease or correct (the quantity or level of) wild-type PSEN1 (or PSEN2) transcripts, and the oligonucleotide is able to normalize, correct, or reverse abnormal A042/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio), preferably wherein the assessment is compared to the level of the same mutant transcript or the same Ap ratio at the onset of the treatment (or to corresponding level of the same Ap ratio in healthy subjects). The assessment may be carried out in a cell, a tissue, plasma, or CSF or subject. When a subject is treated, said ratios are preferably assessed in plasma of said subjects. A subject may be an asymptomatic FAD subject. Alternatively, the subject may be a symptomatic FAD subject. If the subject is a symptomatic FAD subject, the assessment is preferably carried out using plasma. An alternative way of assessing the effect of the oligonucleotide on the Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio) is applying imaging technologies to assess Ap plaque accumulation in the brain of treated subjects. Imaging technology may include PET scans on FAD patients. Imaging technology may also include histology on murine tissues.
In another preferred embodiment, an antisense oligonucleotide of the invention is considered to be active when: - the oligonucleotide is able to preferentially silence, inactivate, breakdown, knock down or reduce or decrease or correct (the quantity or level of) mutated PSEN1 (or PSEN2) proteins,
- the oligonucleotide is not (or minimally) able to silence, inactivate, breakdown, knock down or reduce or decrease or correct (the quantity or level of) wild-type PSEN1 (or PSEN2) proteins, and
- the oligonucleotide is able to normalize, correct, or reverse the abnormal increased soluble A042/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the Ap42/38 and/or Ap42/40 ratios, more preferably the Ap42/38 ratio), preferably wherein the assessment is compared to the level of the same mutant protein orthe same Ap ratio at the onset of the treatment (or to corresponding level of the same Ap ratio in healthy subjects). The assessment may be carried out in a cell, a tissue, plasma, or CSF or subject. A subject may be an asymptomatic FAD subject. Alternatively, the subject may be a symptomatic FAD subject. If the subject is a symptomatic FAD subject, the assessment is preferably carried out using plasma.
The silencing or inactivation or breaking down or reduction or decrease or knocking down of the mutated transcript is expected to induce a similar reduction or decrease of the levels of the mutated protein (preferably PSEN1).
In a more preferred embodiment, an antisense oligonucleotide of the invention may be considered to be active when:
- the oligonucleotide is able to preferentially silence, inactivate, breakdown, knock down or reduce or decrease or correct (the quantity or level of) mutated PSEN1 (or PSEN2) transcripts,
- the oligonucleotide is not (or minimally) able to silence, inactivate, breakdown, knock down or reduce or decrease or correct (the quantity or level of) wild-type PSEN1 (or PSEN2) transcripts,
- the oligonucleotide is able to normalize, correct, or reverse abnormal Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably the A042/38 and/or Ap42/40 ratios, more preferably the A042/38 ratio) and,
- the oligonucleotide is able to reduce the formation of Ap plaques, preferably when compared to the level of the same (mutant) transcript or the same Ap ratio or the quantity of Ap plaques at the onset of the treatment (or to corresponding level of the same transcript, or Ap ratio in healthy subjects).
The A042/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios may be assessed using ELISA, preferably as carried out in the experimental part. The formation of Ap plaques may be assessed using imaging technologies in the brain of treated subjects. Imaging technology may include PET scans on FAD patients. Imaging technology may also include histology on murine tissues.
The assessment may be carried out in a cell, a tissue, plasma, or CSF or subject. When a subject is treated, said ratios are preferably assessed in plasma of said subjects. A subject may be an asymptomatic FAD subject. Alternatively the subject may be a symptomatic FAD subject. If the subject is a symptomatic FAD subject, the assessment is preferably carried out using plasma.
In an embodiment, the antisense oligonucleotide of the invention preferentially targets a mutated allele (and not, or minimally, the WT allele) of protein PSEN1 or PSEN2 when present in a cell expressing said mutated allele. Preferred cells have been earlier defined herein. In a preferred embodiment, the antisense oligonucleotide of the invention preferentially targets a mutated allele (and not, or minimally , the WT allele) of protein PSEN1 (preferably human protein PSEN1) of the y-secretase complex when present in a cell expressing said mutated allele. Preferred cells have been earlier defined herein.
Preferably, an antisense oligonucleotide of the invention is said to be active when,
- it is able to silence, inactivate, knock down, reduce, break down, correct or decrease (the quantity or levels of) said mutated allele or transcript, and
- the level of the corresponding wild-type allele or transcript is not less than 50%, 60%, 70%, 80%, 90%, 95% or 100% of the level of the same allele or transcript at the onset of the treatment.
The allele or transcript is preferably from the PSEN1 or PSEN2 gene, more preferably from the PSEN1 gene. In principle, the invention is not limited to a specific mutation found in PSEN1 or PSEN2. There are already several hundred mutations known for PSENI that are linked with FAD. Examples of mutations in PSEN1 that cause FAD and could be potentially targeted by the oligonucleotide of the invention: R35Q; E69D; A79V; V82L; I83T; M84V; L85P; P88L; V89L; V89L; C92S; V94M; V96F; V97L; T99A; F105C; F105I; F105L; F105V; R108Q; L113P; L113Q; Y1 15C; Y115D; Y115H; T116I; T116N; T116R; P117A; P117L; P117R; P117S; E120D ; E120D ; E120G; E120K; E123K; H131 R; S132A; L134R; N135D; N135S; N135Y; A136G; M139I; M139I; M139K; M139L; M139T; M139V; V142F; I143F; I143M; I143N; I143T; 1143V; M146I; M146I; M146I; M146L; M146L; M146V; T147I; T147P; L150P; LI 53 V; Y154C; Y154N; Y156F; R157inslY; R157S; H163P; H163R; H163Y; A164V; W165C; W165C; W165G; L166H; L166P; L166R; L166V; S169L; S169P; S170F; S170P; L171 P; L173F; L173F; L173W; L174del; L174M; L174R; F175S; F176L; F177L; F177S; S178P; G183V; E184D; E184G; V191A; I202F; G206A; G206D; G206S; G206V; G209A; G209E; G209R; G209V; S212Y; I213F; I213L; I213T; H214D; H214N; H214Y; G217D; G217R; L219F; L219P; L219R; R220P; Q222H; Q222P; Q222R; Q223R; L226F; L226R; I229F; S230I; S230N; S230R; A231 P; A231T; A231V; L232P; M233I ; M233I ; M233L ; M233L ; M233T; M233V; L235P; L235R; L235V; F237I; F237L; 1238M; K239N; T245P; A246E; A246P; L248P; L248R; L250F; L250S; L250V; Y256S; D257A, A260V; V261 F; V261 L; L262F; L262V; C263F; C263R; P264L; G266S; P267A; P267L; P267S; R269G; R269H; L271 V; V272A; E273A; E273G; T274R; A275V; R278I; R278K; R278S; R278T; E280A; E280G; E280K; L282F; L282R; L282V; F283L; P284L; P284S; A285V; L286P; L286V; T291A; T291 P; K311 R; E318G; D333G; R352C; R352_S353insR; T354I; R358Q; S365A; S365Y; R377M; R377W; G378E; G378V; G378fs; L381 F; L381V; G384A; F386I; F386S; F388L; S390I; S390N; V391 F; V391 G; L392P; L392V; G394V; A396T; N405S; I408T; A409T; C410Y; V412I; I416T; G417S; L418F; L420R; L424F; L424H; L424R; L424V; A426P; A431 E; A431 V; A434C; A434T; L435F; P436Q;
P436S; I437V; I439S; I439V; or M457V.
Examples of mutations in PSEN2 that cause FAD and that could be targeted by an oligonucleotide of the invention: T18M; R29H; G34S; R62C; R62H; P69A; R71W; K82R; A85V; V101 M; K115Efs; T122P; T122R; P123L; E126fs; E126K; S130L; V139M; N141 I; N141Y; L143H; V148I; K161 R; R163H; H169N; M174V; S 175C; G212V; V214L; Q228L; Y231 C; 1235F; A237V; L238F; L238P; M239I; M239V; A252T; A258T; T301 M; K306fs; P334A; P334R; P348L; A377V; V393M; T430M; or D439A
In an embodiment, the PSEN1 mutation is selected from the group consisting of the most frequent PSEN1 mutations: P117L, M139T, M139V, M146I, H163R, G206A, P264L, E280A, L392V and A431 E , more preferably E280A and A431 E. Most preferred mutation in PSEN1 is A431 E. In an embodiment, the PSEN1 mutation is selected from the group consisting of the most frequent PSEN1 mutations: P117L, M139T, M139V, M146I, H163R, G206A, P264L, E280A, L392V, A431 E, and S212Y, more preferably E280A and A431 E. Most preferred mutation in PSEN1 is A431 E
In an embodiment, the PSEN1 mutation is selected from the group consisting of the mutations caused by a change of a guanine, thymine or cytosine to an adenosine in the coding sequence resulting in one of the following mutations in the PSEN1 protein: R35Q, V94M, F105I, R108Q, L113Q, T116N, P117T, P117Q, E120K, E123K, M139K, M139I, V142I, M146I, V151 M, Y154N, L166H, L174M, 11 SON, G206S, G206D, G209R, G209E, S212Y, H214N, G217D, S230N, A231T, M233I, F237I, A246E, Y256N, V261 I, G266S, R269H, V272D, T274K, R278K, E280K, R358Q, A360T, S365Y, G378E, F386I, F386L, S390N, A396T, A409T, C410Y, G417S, L424H, A431 E, A434T, P436Q. Most preferred mutation in PSEN1 is A431 E. For all these mutations, the oligonucleotide may be designed as follows: when the base of the nucleotide present in the oligonucleotide and targeting the mutation is a thymine, said thymine is replaced by an uracil. Examples are provided of oligonucleotides targeting the A431 E mutated allele of PSEN1 . Such examples are disclosed in SEQ ID NO: 26, 28, 30, 33, 35, 37, 39. Examples are also provided of oligonucleotides targeting the S212Y mutated allele of PSEN1. Such examples are disclosed in SEQ ID NO: 188, 190, 192, 194, 198, 200.
Preferred oligonucleotides targeting this mutation via a thymine have this thymine be replaced by a uracil and this uracil is at the first, second or third position of their central part. Said preferred oligonucleotides targeting this mutation via a thymine have this thymine replaced by a uracil at the sixth, seventh, or eighth nucleotide in the oligonucleotide sequence starting from 5’ to 3’. Other preferred oligonucleotides targeting this mutation via a thymine have this thymine replaced by a uracil and this uracil is at the first, second, third, or seventh position of the central part of the oligonucleotide. Said preferred oligonucleotides targeting this mutation via a thymine have this thymine replaced by a uracil at the sixth, seventh, eighth, or twelfth nucleotide in the oligonucleotide sequence starting from 5’ to 3’. This replacement increases mutant allele selectivity (See examples 3-4), possibly due to a slightly decreased melting temperature (Tm). Indeed, examples 3 and 4 indicate that antisense oligonucleotides with a thymine-uracil replacement at the above-mentioned preferred nucleotide positions exhibit an increased mutant allele selectivity of PSEN1 .Examples are provided of oligonucleotides targeting the A431 E or S212Y mutated allele of PSEN1. Preferred examples are found in SEQ ID NO: 28, 30, 33, 37, 39, 47, 97, 99, 115, 192, 194, 198.
As used herein, the term “central part” or “central region” of an antisense oligonucleotide refers to a specific section or segment of the oligonucleotide sequence. The precise definition and boundaries of the central part can vary depending on the context of the invention. In context of the first aspect of the invention, the central part may be determined based on the nucleotide position relative to the ends of the oligonucleotide. In context of the second aspect of the invention, the central part refers to the distinct region of the gapmer present between two flanking regions/wings. With respect to both aspects, the skilled person understands that the central part is designed to specifically target a mutated allele of a protein of the y-secretase complex.
As known to the skilled person an oligonucleotide is a polymer of nucleotides and/or a polymer of nucleotides analogues. The expression “nucleotide analogue” may be replaced by “derived from a nucleotide”. An oligonucleotide comprises or consists of repeating monomers. Within the context of the invention, an oligonucleotide comprises up to 50 nucleotides and/or nucleotide analogues. Said oligonucleotide may have 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. Such oligonucleotide may also be identified as an oligonucleotide having from 10 to 50 nucleotides or from 12 to 50 nucleotides. Attractive results were obtained with oligonucleotides having a length ranged from 15 to 30 nucleotides, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.
In an embodiment, the antisense oligonucleotide of the invention comprises nucleotides and/or nucleotides analogues. In another embodiment, an antisense oligonucleotide of the invention comprises 15 to 30 nucleotides including a central region of DNA nucleotides (and/or DNA nucleotide analogues) flanked at each end by wing regions comprising RNA nucleotides analogues. In a preferred embodiment, the antisense oligonucleotide comprising 15 to 30 nucleotides including a central region of DNA nucleotides flanked at each end by wing regions comprising RNA nucleotides analogues relates to the antisense oligonucleotide of the second aspect of the invention. A DNA nucleotide analogue may be present in the central region of DNA nucleotides as long as the natural recruitment of RNaseH to the AON-target RNA duplex is not significantly reduced or hindered or completely abolished and/or its on target activity is not significantly reduced. In this context, “its activity is not significantly reduced” means that the activity of an oligonucleotide with one or more DNA analogues nucleotides in the central part is better or similar or not less than 50%, 60%, 70%, 80%, or 90% compared to oligonucleotide with only DNA nucleotides in the central part. Accordingly, an antisense oligonucleotide as defined herein is able to recruit RNaseH to silence, inactivate, knock down, break down, decrease or reduce (the levels or expression of) the targeted mutant allele.
In another embodiment, an antisense oligonucleotide of the invention comprises 15 to 30 nucleotides including a central region of consecutive DNA nucleotides (and/or DNA nucleotide analogues) flanked at each end by wing regions comprising RNA nucleotides analogues. In another embodiment, an antisense oligonucleotide of the invention comprises 15 to 30 nucleotides including a central region of consecutive DNA nucleotides (and/or DNA nucleotide analogues) flanked at each end by wing regions comprising consecutive RNA nucleotides analogues. In a preferred embodiment, an antisense oligonucleotide of the invention comprises 15 to 30 nucleotides including a central region of 5 to 15 consecutive DNA nucleotides (and/or DNA nucleotide analogues) flanked at each end by wing regions comprising 1 to 5 RNA nucleotide analogues. In a preferred embodiment, an antisense oligonucleotide of the invention comprises 15 to 30 nucleotides including a central region of 5 to 15 consecutive DNA nucleotide analogues flanked in each end by wing regions comprising 1 to 5 RNA nucleotide analogues. In a preferred embodiment, the antisense oligonucleotide comprising 15 to 30 nucleotides including a central region of (consecutive) DNA nucleotides (and/or DNA nucleotide analogues) flanked at each end by wing regions comprising (consecutive) RNA nucleotides analogues relates to the antisense oligonucleotide of the second aspect of the invention. In another preferred embodiment, the antisense oligonucleotide comprising 15 to 30 nucleotides including a central region of 5 to 15 (consecutive) DNA nucleotides (and/or DNA nucleotide analogues) flanked at each end by wing regions comprising 1 to 5 RNA nucleotides analogues relates to the antisense oligonucleotide of the second aspect of the invention.
In an embodiment, the central region comprises 5, 6, 7, 8, 9 , 10, 11 , 12, 13, 14 or 15 DNA nucleotides and/or DNA nucleotide analogues. Preferably, the length of the central region is 9, 10 or 11 DNA nucleotides and/or DNA nucleotide analogues. In another embodiment, one wing region comprises 1 , 2, 3, 4 or 5 RNA nucleotide analogues. Preferably, the length of one wing region is 2 or 4 RNA nucleotide analogues. In another embodiment, each wing region comprises 1 , 2, 3, 4 or 5 RNA nucleotide analogues. Preferably, the length of each wing region is 2 or 5 RNA nucleotide analogues. It is also encompassed by the invention that each wing region is not identical in length and/or in the type of chemistry used in the RNA nucleotide analogues.
In an embodiment, the central region comprises 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 consecutive DNA nucleotides and/or DNA nucleotide analogues. Preferably, the length of the central region is 9, 10 or 11 consecutive DNA nucleotides and/or DNA nucleotide analogues. In another embodiment, one wing region comprises 1 , 2, 3, 4 or 5 consecutive RNA nucleotide analogues. Preferably, the length of one wing region is 2 or 5 consecutive RNA nucleotide analogues. In an embodiment, each wing region comprises 1 , 2, 3, 4 or 5 consecutive RNA nucleotide analogues. Preferably, the length of each wing region is 2 or 5 consecutive RNA nucleotide analogues. It is also encompassed by the invention that each wing region is not identical in length and/or in the type chemistry used in the RNA nucleotide analogues.
In an embodiment, the central region is 10 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 5 consecutive RNA nucleotide analogues. In another embodiment, the central region is 10 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 4 consecutive RNA nucleotide analogues. In another embodiment, the central region is 10 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 3 consecutive RNA nucleotide analogues. In another embodiment, the central region is 10 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 2 consecutive RNA nucleotide analogues. In another embodiment, the central region is 10 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 1 consecutive RNA nucleotide analogue.
In an embodiment, the central region is 11 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 5 consecutive RNA nucleotide analogues, In another embodiment, the central region is 11 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 4 consecutive RNA nucleotide analogues, In another embodiment, the central region is 11 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 3 consecutive RNA nucleotide analogues, In another embodiment, the central region is 11 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 2 consecutive RNA nucleotide analogues, In another embodiment, the central region is 11 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 1 consecutive RNA nucleotide analogue.
In an embodiment, the central region is 9 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 5 consecutive RNA nucleotide analogues. In another embodiment, the central region is 9 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 4 consecutive RNA nucleotide analogues. In another embodiment, the central region is 9 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 3 consecutive RNA nucleotide analogues. In another embodiment, the central region is 9 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 2 consecutive RNA nucleotide analogues. In another embodiment, the central region is 9 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 1 consecutive RNA nucleotide analogue.
In an embodiment, the central region is 12 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 5 consecutive RNA nucleotide analogues. In another embodiment, the central region is 12 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 4 consecutive RNA nucleotide analogues. In another embodiment, the central region is 12 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 3 consecutive RNA nucleotide analogues. In another embodiment, the central region is 12 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 2 consecutive RNA nucleotide analogues. In another embodiment, the central region is 12 consecutive DNA nucleotides and/or DNA nucleotide analogues, and each wing region is 1 consecutive RNA nucleotide analogue.
In a preferred embodiment, the mutation in the allele or transcript of PSEN1 or PSEN2 is targeted by a nucleotide of the oligonucleotide being present in the central part of the oligonucleotide; the central part being 5 to 15 nucleotides and each wing being 1 to 5 nucleotides. In a preferred embodiment, the antisense oligonucleotide comprising a nucleotide, present in the central part of the oligonucleotide, which targets the mutation in the allele or transcript of PSEN1 or PSEN2 relates to the antisense oligonucleotide of the second aspect of the invention. It was surprisingly found that the efficacy and selectivity of the oligonucleotide for the mutated PSEN1 or PSEN2 allele or transcript is optimal when the nucleotide of the oligonucleotide targeting the mutated nucleotide of PSEN1 or PSEN2 is located in the central part of the oligonucleotide. In particular, as shown in Examples 3 and 4, antisense oligonucleotides have an increased selectivity towards the mutant PSEN1 allele when the nucleotide targeting the mutated nucleotide of PSEN1 is located within the first five nucleotides of the central part of the oligonucleotide starting from 5’ to 3’ (i.e;, nucleotides located at position 6, 7, 8, 9, or 10 in the oligonucleotide sequence starting from 5’ to 3’).
In an embodiment, said nucleotide is the first, second, third, fourth, fifth, sixth, seventh, eighth, nineth, tenth, eleventh or twelfth nucleotide in the central part of the oligonucleotide starting from 5’ to 3’. In said embodiment, said nucleotide is the sixth, seventh, eighth, nineth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, or eighteenth nucleotide in the oligonucleotide sequence starting from 5’ to 3’. In a preferred embodiment, said nucleotide is the first, second, third, fourth, or fifth nucleotide in the central part of the oligonucleotide starting from 5’ to 3’. Accordingly, in said preferred embodiment, said nucleotide is the sixth, seventh, eighth, nineth, or tenth nucleotide in the oligonucleotide sequence starting from 5’ to 3’. In an embodiment, said nucleotide is the first, second, third, or fifth nucleotide in the central part of the oligonucleotide starting from 5’ to 3’. In said preferred embodiment, said nucleotide is the sixth, seventh, eight, or tenth nucleotide in the oligonucleotide sequence part starting from 5’ to 3’. In an embodiment, when the base of said nucleotide present in the oligonucleotide and targeting the mutation is a thymine, said thymine is replaced by an uracil. Examples are provided of oligonucleotides targeting the A431 E mutated allele of PSEN1 . Such examples are disclosed in SEQ ID NO: 26, 28, 30, 33, 35, 37, 39.
Examples are also provided of oligonucleotides targeting the S212Y mutated allele of PSEN1. Such examples are disclosed in SEQ ID NO: 188, 190, 192, 194, 198, 200. Preferred oligonucleotides targeting this mutation via a thymine have this thymine be replaced by an uracil and this uracil is at the first, second or third position of their central part. Accordingly, preferred oligonucleotides targeting this mutation have the thymine replaced by an uracil at the sixth, seventh, or eight position of the oligonucleotide sequence starting from 5’ to 3’. Other preferred oligonucleotides targeting this mutation via a thymine have this thymine be replaced by an uracil and this uracil is at the first, second, third, or seventh position of the central part of the oligonucleotide. Said preferred oligonucleotides have the thymine replaced by an uracil at the sixth, seventh, eighth, or twelfth position of the oligonucleotide sequence starting from 5’ to 3’. This replacement increases mutant allele selectivity, possibly due to a slight decreased Tm. Preferred examples are found in SEQ ID NO: 28, 30, 33, 37, 39, 47, 97, 99, 115, 192, 194, 198.
Therefore, in a preferred embodiment, the oligonucleotide is as follows: it comprises a central part of 5 to 15 nucleotides and two wings, each of 1 to 5 nucleotides and the mutation present in the mutated allele or transcript of PSEN1 or PSEN2 is targeted by the first, second, third, fourth, fifth, sixth, seventh, eighth, nineth, tenth, eleventh or twelfth nucleotide of the central part of the oligonucleotide.
In a preferred embodiment, the oligonucleotide is as follows: it comprises a central part of 5 to 15 nucleotides and two wings, each of 1 to 5 nucleotides and the mutation present in the mutated allele or transcript of PSEN1 or PSEN2 is targeted by the sixth, seventh, eighth, nineth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, or eighteenth nucleotide in the oligonucleotide sequence starting from 5’ to 3’.
Accordingly, in a preferred embodiment, the antisense oligonucleotide relates to the antisense oligonucleotide of the second aspect of the invention.
In another preferred embodiment, the base of the nucleotide of the oligonucleotide targeting the mutation present in the PSEN1 or PSEN2 allele or transcript is a RNA base. In a more preferred embodiment, said RNA base is a uracil. More preferably, the target mutation is a C to A mutations, but also the target mutation might be a G or T to A mutations.
In an embodiment, the antisense oligonucleotide of the invention comprises nucleotides and nucleotide analogues. Nucleotides may be RNA or DNA nucleotides. The most common naturally occurring nucleotides in RNA are adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, thymidine monophosphate, and uridine monophosphate. These consist of a pentose sugar ribose, a 5’-linked phosphate group which is linked via a phosphate ester, and a T-linked base. The same holds for DNA wherein deoxyribose is present instead of ribose. The sugar connects the base and the phosphate linkage, and is therefore often referred to as the scaffold of the nucleotide. A modification in the pentose sugar is therefore often referred to as a scaffold modification. A sugar modification may therefore be called a scaffold modification. For several modifications, the original pentose sugar may be replaced in its entirety by another moiety that similarly connects the base and the phosphate. It is therefore understood that while a pentose sugar is often a scaffold, a scaffold is not necessarily a pentose sugar.
A base, sometimes called a nucleobase, is generally adenine, cytosine, guanine, thymine, or uracil, or a derivative thereof. Cytosine, thymine, and uracil are pyrimidine bases, and are generally linked to the scaffold through their 1 -nitrogen. Adenine and guanine are purine bases, and are generally linked to the scaffold through their 9-nitrogen. A base present in an oligonucleotide is usually complementary to the target region. If such a base is a modified base or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces. “Base pairing” refers to the binding of two bases (or nucleobases) to each other by hydrogen bonds. Specifically, a nucleobase analogue replacing cytosine is capable of base pairing with guanine, a nucleobase analogue replacing guanine is capable of base pairing with cytosine, a nucleobase analogue replacing adenine is capable of base pairing with uracil and a nucleobase replacing uracil is capable of base pairing with adenine.
A nucleotide is generally connected to neighboring nucleotides through condensation of its 5’- phosphate moiety to the 3’-hydroxyl moiety of the neighboring nucleotide monomer. Similarly, its 3’-hydroxyl moiety is generally connected to the 5’-phosphate of a neighboring nucleotide monomer. This forms phosphodiester bonds. The phosphodiesters and the scaffold form an alternating copolymer. The bases are grafted to this copolymer, namely to the scaffold moieties. Because of this characteristic, the alternating copolymer formed by linked monomers of an oligonucleotide is often called the backbone of the oligonucleotide. Because the phosphodiester bonds connect neighboring monomers together, they are often referred to as backbone linkages. It is understood that when a phosphate group is modified so that it is instead an analogous moiety such as a phosphorothioate (PS), such a moiety is still referred to as the backbone linkage of the monomer. This is referred to as a backbone linkage modification. In general terms, the backbone of an oligonucleotide is thus comprised of alternating scaffolds and backbone linkages. In an embodiment, the backbone of the oligonucleotide (central part and the wings) is fully modified, preferably being a PS backbone.
In an embodiment the antisense oligonucleotide comprises a central part comprising DNA nucleotides (or DNA nucleotides analogues) and modified RNA parts in each wing. In a preferred embodiment, the antisense oligonucleotide comprising a central part comprising DNA nucleotides (or DNA nucleotides analogues) and modified RNA parts in each wing relates to the antisense oligonucleotide of the second aspect of the invention. A RNA or DNA nucleotide may be modified and may therefore be considered as an RNA or DNA nucleotide analogue when it comprises a modified base and/or a modified sugar. In addition, the internucleoside linkage linking two adjacent nucleosides may be modified compared to the phosphodiester bond linking two nucleosides. A “modified internucleoside linkage” may be replaced by the wording “backbone linkage modification” as explained earlier herein.
In an embodiment, the antisense oligonucleotide of the invention comprises a DNA central part (or region) flanked at each end (wing) by modified RNA parts (regions). In another embodiment, the antisense oligonucleotide of the invention comprises a modified DNA central part (or region) flanked at each end (wing) by modified RNA parts (regions). In a preferred embodiment, the antisense oligonucleotide of the invention comprising a modified DNA central part flanked at each end by modified RNA parts relates to the antisense oligonucleotide of the second aspect of the invention.
In an embodiment, the oligonucleotide of the invention, consisting of a 5’ wing, a 3’ wing, and a central part (gap) is single-stranded. Such structure (a 5’ wing, a 3’ wing, and a central part (gap)) may also be called gapmer. In a preferred embodiment, the single-stranded antisense oligonucleotide consisting of a 5’ wing, a 3’ wing, and a central part relates to the antisense oligonucleotide of the second aspect of the invention. Said structure is attractive as it allows the oligonucleotide to bind towards target mRNA and to recruit RNaseH. The recruited RNaseH has at least one of the following effects on the targeted mutant allele: to silence, inactivate, knock down, break down, decrease or reduce its levels. In contrast, double-stranded siRNAs are not able to recruit RNaseH but rather depend on Argonaute proteins for silencing target mRNA. Double-stranded siRNAs are loaded into the RNA-induced silencing complex (RISC). Upon RISC loading, the less thermodynamically stable 5'-end of the siRNA is incorporated and guides the RISC to the complementary target mRNA. The mRNA target dissociates from the intact siRNA after Argonaute-dependent cleavage, freeing RISC to regenerate and cleave additional mRNA targets.
The presence of the wings provides the antisense oligonucleotide stability and resistance properties to degradation by exonucleases. Implementation of chemical modifications in the gap and/or wings, comprising base, scaffold, and/or linkage modifications, may improve safety, biodistribution, stability, cellular uptake, intracellular trafficking, target binding affinity, duplex stability and the efficiency of the oligonucleotide of the invention compared to an oligonucleotide consisting of non-modified DNA and/or non-modified RNA analogues. This effect may be at least due to the presence of the modified RNA wings and/or the modified DNA central part. Finetuning of DNA and/or RNA base, scaffold, and/or linkage modifications at specific positions in the oligonucleotide (precision chemistry) may result in oligonucleotides with most favorable characteristics for clinical application.
In an embodiment, the central part/region (gap) of the antisense oligonucleotide comprises DNA nucleotides or DNA nucleotide analogues. In a preferred embodiment, the base and sugar of this part of the antisense oligonucleotide are not modified. However, in a more preferred embodiment, an internucleoside linkage (or the whole backbone) in this central part is modified. In an embodiment, the modified internucleoside linkage is a phosphorothioate or a phosphoramidate internucleoside linkage. A nucleotide of the central part/region of said antisense oligonucleotide may have at least one internucleoside linkage modification and/or at least one base modification compared to an antisense oligonucleotide with a full non-modified DNA central part.
In an embodiment, a wing of the antisense oligonucleotide comprises modified RNA nucleotides and/or modified internucleoside linkages. In an embodiment, both wings of the antisense oligonucleotide comprise modified RNA nucleotides and/or modified internucleoside linkages. A wing of said antisense oligonucleotide may have at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to a nonmodified RNA-based antisense oligonucleotide. In an embodiment, the modified internucleoside linkage is a phosphorothioate or a phosphoramidate internucleoside linkage.
A base modification in the central part and/or in the wing of the oligonucleotide includes a modified version of the natural purine and pyrimidine bases (e.g. adenine, uracil, guanine, cytosine, and thymine), such as hypoxanthine (e.g. inosine), orotic acid, agmatidine, lysidine, pseudouracil, 2- thiopyrimidine (e.g. 2-thiouracil, 2-thiothymine), G-clamp and its derivatives, 5-substituted pyrimidine (e.g. 5-halouracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5- hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, Super T), 7- deazaguanine, 7-deazaadenine, 2,6-diaminopurine, 7-aza-2,6-diaminopurine, 8-aza-7- deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, Super G, Super A, and N4-ethylcytosine, or derivatives thereof; N2-cyclopentylguanine (cPent-G), N2-cyclopentyl-2- aminopurine (cPent-AP), and N2-propyl-2-aminopurine (Pr-AP), or derivatives thereof; and degenerate or universal bases, like 2,6-difluorotoluene or absent bases like abasic sites (e.g. 1- deoxyribose, 1 ,2-dideoxyribose, 1-deoxy-2-0-methylribose; or pyrrolidine derivatives in which the ring oxygen has been replaced with nitrogen (azaribose)). Examples of derivatives of Super A, Super G and Super T can be found in US patent 6,683,173 (Epoch Biosciences), which is incorporated here entirely by reference. cPent-G, cPent-AP and Pr-AP were shown to reduce immunostimulatory effects when incorporated in siRNA (Peacock H. et al.).
A preferred modified base is 5-methylcytosine.
Depending on the length of each wing, an antisense oligonucleotide of the invention may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 base modifications. It is also encompassed by the invention to introduce more than one distinct base modification in said wing of said antisense oligonucleotide.
Depending on the length of the central part, an antisense oligonucleotide of the invention may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 base modifications. It is also encompassed by the invention to introduce more than one distinct base modification in the central part of said antisense oligonucleotide.
Depending on the length of the antisense oligonucleotide, an antisense oligonucleotide of the invention may comprise at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more base modifications. It is also encompassed by the invention to introduce more than one distinct base modification in said antisense oligonucleotide.
A modified sugar in a nucleotide of a wing and/or in the central part of the antisense oligonucleotide is synonymous of a scaffold modification of the oligonucleotide.
A scaffold modification can include a modified version of the ribosyl moiety, such as 2’- O-modified RNA such as 2’-O-alkyl or 2’-O-(substituted)alkyl e.g. 2’-O-methyl, 2’-O-(2- cyanoethyl), 2’-0-(2-methoxy)ethyl (2’-MOE), 2’-0-(2-thiomethyl)ethyl, 2’-0-butyryl, 2’-O- propargyl, 2’-O-acetalester (such as e.g. Biscans et al. Bioorg. Med. Chem. 2015, 23, 5360), 2’- O-allyl, 2’-0-(2S-methoxypropyl), 2’-0-(/V-(aminoethyl)carbamoyl)methyl) (2’-AECM), 2’-O-(2- carboxyethyl) and carbamoyl derivatives (Yamada et al. Org. Biomol. Chem. 2014, 12, 6457), 2’- 0-(2-amino)propyl, 2’-0-(2-(dimethylamino)propyl), 2’-0-(2-amino)ethyl, 2’-O-(2- (dimethylamino)ethyl); 2’-deoxy (DNA); 2’-0-(haloalkoxy)methyl (Arai K. et al. Bioorg. Med. Chem. 2011 , 21, 6285) e.g. 2’-0-(2-chloroethoxy)methyl (MCEM), 2’-O-(2,2- dichloroethoxy)methyl (DCEM); 2’-0-alkoxycarbonyl e.g. 2’-0-[2-(methoxycarbonyl)ethyl] (MOCE), 2’-0-[2-(/V-methylcarbamoyl)ethyl] (MCE), 2’-0-[2-(/V,/V-dimethylcarbamoyl)ethyl] (DCME), 2’-0-[2-(methylthio)ethyl] (2’-MTE), 2’-(w-0-serinol); 2’-halo e.g. 2’-F, FANA (2’-F arabinosyl nucleic acid); 2’,4’-difluoro-2’-deoxy; carbasugar and azasugar modifications; 3’-O- substituted e.g. 3’-O-methyl, 3’-0-butyryl, 3’-0-propargyl; 4’-substituted e.g. 4’-aminomethyl-2’- O-methyl or 4’-aminomethyl-2’-fluoro; 5’-subtituted e.g. 5’-methyl or CNA (0stergaard et al. ACS Chem. Biol. 2014, 22, 6227); and their derivatives.
A scaffold modification can include a bicyclic nucleic acid monomer (BNA) which may be a bridged nucleic acid monomer. Each occurrence of said BNA may result in a monomer that is independently chosen from the group consisting of a conformationally restricted nucleotide (CRN) monomer, a locked nucleic acid (LNA) monomer, a xylo-LNA monomer, an a-LNA monomer, an a-L-LNA monomer, a p-D-LNA monomer, a 2’-amino-LNA monomer, a 2’-(alkylamino)-LNA monomer, a 2’-(acylamino)-LNA monomer, a 2’-/V-substituted-2’-amino-LNA monomer, a 2’-thio- LNA monomer, a (2’-O,4’-C) constrained ethyl (cEt) BNA monomer, a (2’-O,4’-C) constrained methoxyethyl (cMOE) BNA monomer, a 2’,4’-BNANC(N-H) monomer, a 2’,4’-BNANC(N-Me) monomer, a 2’,4’-BNANC(N-Bn) monomer, an ethylene-bridged nucleic acid (ENA) monomer, a carba LNA (cLNA) monomer, a 3,4-dihydro-2/7-pyran nucleic acid (DpNA) monomer, a 2’-C- bridged bicyclic nucleotide (CBBN) monomer, a heterocyclic-bridged BNA monomer (such as triazolyl or tetrazolyl-linked), an amido-bridged BNA monomer, an urea-bridged BNA monomer, a sulfonamide-bridged BNA monomer, a bicyclic carbocyclic nucleotide monomer, a TriNA monomer, an a-L-TriNA monomer, a bicyclo DNA (bcDNA) monomer, an abcDNA monomer, an F-bcDNA monomer, a tricyclo DNA (tcDNA) monomer, an F-tcDNA monomer, an oxetane nucleotide monomer, a locked PMO monomer derived from 2’-amino-LNA, a guanidine-bridged nucleic acid (GuNA) monomer, a spirocyclopropylene-bridged nucleic acid (scpBNA) monomer, and derivatives thereof.
A preferred sugar modification is selected from: - 2’-0-modified RNA, more preferably 2’-0-alkyl or 2’-0-(substituted)alkyl, even more preferably 2’-O-methyl (2’-OMe) or 2’-0-(2-methoxy)ethyl (2’-MOE)
- (BNA), more preferably a (CRN) monomer or a locked nucleic acid (LNA) monomer.
More preferred sugar modifications are 2’-OMe, 2’-MOE, and a locked nucleic acid (LNA). Depending on the length of each wing, an antisense oligonucleotide of the invention may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 sugar modifications. It is also encompassed by the invention to introduce more than one distinct sugar modification in said wing of said antisense oligonucleotide.
Depending on the length of the central part, an antisense oligonucleotide of the invention may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 sugar modifications. It is also encompassed by the invention to introduce more than one distinct sugar modification in said wing of said antisense oligonucleotide.
Depending on the length of the antisense oligonucleotide, an antisense oligonucleotide of the invention may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more sugar modifications. It is also encompassed by the invention to introduce more than one distinct sugar modification in said antisense oligonucleotide.
Antisense oligonucleotides according to the invention can comprise backbone linkage modifications in their wing and/or in their central part. A backbone linkage modification can be, but is not limited to, a modified version of the phosphodiester present in RNA, such as phosphorothioate (PS), chirally pure phosphorothioate, (R)-phosphorothioate, (S)- phopshorothioate, phosphorodithioate (PS2), phosphonoacetate (PACE), phosphonoacetamide (PACA), thiophosphonoacetate (thioPACE), thiophosphonoacetamide, phosphorothioate prodrug, H-phosphonate, methyl phosphonate, methyl phosphonothioate, methyl phosphate, methyl phosphorothioate, ethyl phosphate, ethyl phosphorothioate, boranophosphate, boranophosphorothioate, methyl boranophosphate, methyl boranophosphorothioate, methyl boranophosphonate, methyl boranophosphonothioate, phosphate, phosphotriester, aminoalkylphosphotriester, and their derivatives. Another modification includes phosphoryl guanidines, acylphosphoramidates, sulfonylphosphoramidates, phosphoramidite, phosphoramidate, N3’->P5’ phosphoramidate, phosphordiamidate, phosphorothiodiamidate, sulfamate, dimethylenesulfoxide, amide, sulfonate, siloxane, sulfide, sulfone, formacetyl, thioformacetyl, methylene formacetyl, alkenyl, methylenehydrazino, sulfonamide, amide, triazole, oxalyl, carbamate, methyleneimino (MMI), and thioacetamido nucleic acid (TANA); and their derivatives. Examples of chirally pure phosphorothioate linkages are described in e.g. WO2014/010250 or WO2017/062862 (WaVe Life Sciences). Examples of phosphoryl guanidine linkages are described in WO2016/028187 (Noogen). Various salts, mixed salts and free acid forms are also included, as well as 3’->3’ and 2’->5’ linkages.
Preferred backbone linkage modifications are phosphorothioate and phosphoramidate.
More preferred backbone linkage modifications are phosphorothioate and phosphoramidate (preferably a mesyl-phosphoramidate). Depending on its length, an antisense oligonucleotide of the invention may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or more backbone linkage modifications. It is also encompassed by the invention to introduce more than one distinct backbone modification in said antisense oligonucleotide.
In an embodiment, the antisense oligonucleotide is such that a DNA nucleotide in its central part is not modified.
In another embodiment, the antisense oligonucleotide is such that a DNA nucleotide in its central part is modified and that at least part of the backbone (i.e. internucleoside linkage) in its central part comprises phosphorothioate and/or phosphoramidate (preferably a mesyl- phosphoramidate).
In an embodiment, the antisense oligonucleotide is such that the RNA nucleotide analogues in each of its wings have been modified comprising a modified internucleoside linkage (preferably a phosphorothioate and/or phosphoramidite (preferably a mesyl-phosphoramidate)) and/or a modified sugar (preferably a 2’-MOE, 2’-OMe and/or a locked nucleic acid (LNA) monomer) and/or a modified base (preferably a 5-methylcytosine).
In an embodiment, the antisense oligonucleotide is such that:
• the DNA nucleotides in its central part have not been modified and
• the RNA nucleotide analogues in each of its wings have been modified comprising a modified internucleoside linkage (preferably a phosphorothioate or phosphoramidate (more preferably a mesyl-phosphoramidate) and/or a modified sugar (preferably a 2’-MOE, 2’-OMe and/or a locked nucleic acid (LNA) monomer) and/or a modified base (preferably a 5-methylcytosine).
In an embodiment, the antisense oligonucleotide is such that:
• a DNA nucleotide in its central part has been modified and that the backbone (i.e. at least one internucleoside linkage) in its central comprises phosphorothioate or phosphoramidate (more preferably a mesyl-phosphoramidate) and
• the RNA nucleotide analogues in each of its wings have been modified comprising a modified internucleoside linkage (preferably a phosphorothioate or phosphoramidate (more preferably a mesyl-phosphoramidate) and/or a modified sugar (preferably a 2’-MOE, 2’-OMe and/or a locked nucleic acid (LNA) monomer) and/or a modified base (preferably a 5-methylcytosine).
In an embodiment, the antisense oligonucleotide comprises 15 to 30 or 20 to 30 nucleotides including a central region of 5 to 15 consecutive DNA nucleotides flanked at each end by wing regions comprising 1 to 5 RNA nucleotides analogues. Preferably, the backbone of the oligonucleotide is fully modified (central region and each wing). More preferably the backbone is phosphorothioate and/or phosphoramidite (more preferably mesyl-phosphoramidate).
In an embodiment, the antisense oligonucleotide is as follows:
1 . its DNA nucleotides in its central part have not been modified and the RNA nucleotide analogues in each of its wings have been modified comprising a modified internucleoside linkage (preferably a phosphorothioate or phosphoramidite (more preferably mesyl-phosphoramidate)) and/or a modified sugar (preferably a 2’- MOE, 2’-OMe and/or a locked nucleic acid (LNA) monomer) and/or a modified base (preferably a 5-methylcytosine), or
1 . a DNA nucleotide in its central part has been modified and that the backbone (i.e. at least one internucleoside linkage) in its central part comprises phosphorothioate and/or phosphoramidate (more preferably mesyl-phosphoramidate) and the RNA nucleotide analogues in each of its wings have been modified comprising a modified internucleoside linkage (preferably a phosphorothioate or phosphoramidate (more preferably mesyl-phosphoramidate) and/or a modified sugar (preferably a 2’- MOE, 2’-OMe and/or a locked nucleic acid (LNA) monomer) and/or a modified base (preferably a 5-methylcytosine).
In an embodiment, the antisense oligonucleotide is as follows:
1 . its DNA nucleotides in its central part have not been modified and the RNA nucleotide analogues in each of its wings have been modified comprising a modified internucleoside linkage (preferably a phosphorothioateand/or phosphoramidite (more preferably mesyl-phosphoramidate)) and/or a modified sugar (preferably a 2’-MOE and/or a modified base (preferably a 5-methylcytosine), or
2. a DNA nucleotide in its central part has been modified and that the backbone (i.e. at least one internucleoside linkage) in its central part has been modified comprises a phosphorothioate and/or phosphoramidate (more preferably mesyl-phosphoramidate) and the RNA nucleotide analogues in each of its wings have been modified comprising a modified internucleoside linkage (preferably a phosphorothioate and/or phosphoramidite (more preferably mesyl-phosphoramidate)) and/or a modified sugar (preferably a 2’-MOE and/or a modified base (preferably a 5-methylcytosine).
In a preferred embodiment, the antisense oligonucleotide is as follows: its DNA nucleotides in its central part have been modified and its backbone (i.e. at least one internucleoside linkage) in its central part comprises phosphorothioate linkages, and the RNA nucleotide analogues in each of its wings have been modified comprising a modified backbone which is phosphorothioate linkages, and preferably at least one modified sugar (preferably a 2’-MOE) and/or at least one modified base (preferably a 5-methylcytosine).
In an embodiment, the invention provides human antisense oligonucleotides, which preferentially target a mutated allele of a human protein of the y-secretase pathway, preferably the human protein of the y-secretase pathway is PSEN1. In this embodiment, the antisense oligonucleotide targets, hybridizes, binds and/or is reverse complementary to a mutated allele of a human protein of the y- secretase pathway, preferably PSEN1.
In an embodiment, the base sequence of the oligonucleotide comprises any of SEQ ID NO:1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,
58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,
106, 108, 110, 1 12, 1 14, 116, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,
200, 202, 204, 219, 221 , 223, 225, 227, 229, 231 , 233, 235, 237, 239, 241 , 243, 245, 247, 249,
251 , 253, 255, 257, 259, 261 , 263, 265, 267, 269, 271 , 273, 275, 277. More preferred oligonucleotides comprising one of these base sequences are disclosed below.
In an embodiment the antisense oligonucleotide comprises the following base sequence: TCTTTCTTGAAAATGGCAAG (AON1) (SEQ ID NO:1)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:1. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:1 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:1 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:1 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:1 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:1 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:1 is represented by SEQ ID NO:2: eTseC*seTseTseTsdCsdTsdTsdGsdAsdAsdAsdAsdTsdGseGseC*seAseAseG (SEQ ID NO:2) wherein A means adenosine, G means guanine, T means thymine, C means cytosine, C* means 5-methylcytosine, U means Uracil, e means 2’-MOE, d means DNA, subscript s means a PS internucleoside linkage, subscript o means PO linkage, subscript PNms means PNms linkage, and subscript PNdmi means PNdmi linkage.
This nomenclature is used for all oligonucleotides of the present application, unless otherwise indicated.
In an embodiment the antisense oligonucleotide comprises the following base sequence: ATTCTTTCTTGAAAATGGCA (AON2) (SEQ ID NO:3)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:3. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:3 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:3 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:3 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:3 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:3 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:3 is represented by SEQ ID NO:4: eAseTseTseC*seTsdTsdTsdCsdTsdTsdGsdAsdAsdAsdAseTseGseGseC*seA (SEQ ID NO:4)
In an embodiment the antisense oligonucleotide comprises the following base sequence: CAATTCTTTCTTGAAAATGG (AON3) (SEQ ID NO:5)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:5. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:5 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:5 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate) . More preferably, the oligonucleotide comprising SEQ ID NO:5 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:5 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:5 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:5 is represented by SEQ ID NO:6: eC*seAseAseTseTsdCsdTsdTsdTsdCsdTsdTsdGsdAsdAseAseAseTseGseG (SEQ ID NO:6)
In an embodiment the antisense oligonucleotide comprises the following base sequence:
(AON4) GCAATTCTTTCTTGAAAATG (SEQ ID NO:7)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:7. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:7 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:7 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NOT is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NOT has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:7 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:7 is represented by SEQ ID NO:8: eGseC*seAseAseTsdTsdCsdTsdTsdTsdCsdTsdTsdGsdAseAseAseAseTseG (SEQ ID NO:8)
In an embodiment the antisense oligonucleotide comprises the following base sequence:
(AON5) GGCAATTCTTTCTTGAAAAT (SEQ ID NO:9)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:9. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:9 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:9 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate) . More preferably, the oligonucleotide comprising SEQ ID NO:9 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:9 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:9 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:9 is represented by SEQ ID NQ:10: eGseGseC*seAseAsdTsdTsdCsdTsdTsdTsdCsdTsdTsdGseAseAseAseAseT (SEQ ID NQ:10)
In an embodiment the antisense oligonucleotide comprises the following base sequence:
(AON6) TGGCAATTCTTTCTTGAAAA (SEQ ID NO:11)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:11 . The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:11 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:11 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate) . More preferably, the oligonucleotide comprising SEQ ID NO:11 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:11 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:11 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:11 is represented by SEQ ID NO:12: eTseGseGseC*seAsdAsdTsdTsdCsdTsdTsdTsdCsdTsdTseGseAseAseAseA (SEQ ID NO:12)
In an embodiment the antisense oligonucleotide comprises the following base sequence:
(AON7) CTGGCAATTCTTTCTTGAAA (SEQ ID NO: 13)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:13. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO: 13 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:13 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate ). More preferably, the oligonucleotide comprising SEQ ID NO:13 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:13 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:13 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:13 is represented by SEQ ID NO:14:
Figure imgf000039_0001
In an embodiment the antisense oligonucleotide comprises the following base sequence:
(AON8) AGCTGGCAATTCTTTCTTGA (SEQ ID NO: 15)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:15. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO: 15 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:15 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:15 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:15 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:15 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:15 is represented by SEQ ID NO:16: eAseGseC*seTseGsdGsdCsdAsdAsdTsdTsdCsdTsdTsdTseC*seTseTseGseA (SEQ ID NO:16)
In an embodiment the antisense oligonucleotide comprises the following base sequence:
(AON9) AGAGCTGGCAATTCTTTCTT (SEQ ID NO: 17)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:17. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO: 17 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:17 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 17 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:17 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:17 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:17 is represented by SEQ ID NO:18: eAseGseAseGseC*sdTsdGsdGsdCsdAsdAsdTsdTsdCsdTseTseTseC*seTseT (SEQ ID NO:18)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GAAGAGCTGGCAATTCTTTC (AQN10) (SEQ ID NO:19)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:19. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO: 19 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:19 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate) . More preferably, the oligonucleotide comprising SEQ ID NO:19 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:19 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:19 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:19 is represented by SEQ ID NQ:20: eGseAseAseGseAsdGsdCsdTsdGsdGsdCsdAsdAsdTsdTseC*seTseTseTseC*
(SEQ ID NQ:20)
In an embodiment the antisense oligonucleotide comprises the following base sequence: (AON11) TGGAAGAGCTGGCAATTCTT (SEQ ID NO:21)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:21 . The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:21 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:21 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate) . More preferably, the oligonucleotide comprising SEQ ID NO:21 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:21 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:21 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:21 is represented by SEQ ID NO:22:
Figure imgf000041_0001
In an embodiment the antisense oligonucleotide comprises the following base sequence: ATTGGAAGAGCTGGCAATTC (AON 12) (SEQ ID NO:23)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:23. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:23 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:23 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:23 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:23 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:23 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:23 is represented by SEQ ID NO:24: eAseTseTseGseGsdAsdAsdGscLAsdGsdCsdTsdGsdGsdCseAseAseTseTseC* (SEQ ID NO:24)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON13) (SEQ ID NO:25)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:25. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:25 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:25 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:25 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:25 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:25 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:25 is represented by SEQ ID NO:26: eGseC*seAseAseTsdUsdCsdTsdTsdTsdCsdTsdTsdGsdAseAseAseAseTseG (SEQ ID NO:26)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON14) (SEQ ID NO:27)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:27. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:27 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:27 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:27 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:27 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:27 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:27 is represented by SEQ ID NO:28: eGseGseC*seAseAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdGseAseAseAseAseT (SEQ ID NO:28)
In an embodiment the antisense oligonucleotide comprises the following base sequence:
TGGCAATUCTTTCTTGAAAA (AON15) (SEQ ID NO:29)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:29. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces. In an embodiment, at least one C and preferably all Cs of the oligonucleotide comprising SEQ ID NO:29 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:29 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:29 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:29 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:29 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:29 is represented by SEQ ID NQ:30: eTseGseGseC*seAsdAsdTsdUsdCsdTsdTsdTsdCsdTsdTseGseAseAseAseA (SEQ ID NQ:30)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON16) (SEQ ID NO: 32)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:32. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:32 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:32 present in the gap of said oligonucleotide (C at position 8 and at position 12 of SEQ ID NO:32) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:32 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:32 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:32 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, , the oligonucleotide comprising SEQ ID NO:32 is fully modified with a PS backbone.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate. In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:32 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:32 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:32 is represented by SEQ ID NO:33: eGseGoeC*oeAoeAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdGseAoeAoeAoeAseT (SEQ ID NO:33)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON17) (SEQ ID NO: 34)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:34. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:34 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:32 present in the gap of said oligonucleotide (C at position 8 and at position 12 of SEQ ID NO:34) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:34 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:34 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:34 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NO:34 is fully modified with a PS backbone.
In an embodiment, the oligonucleotide comprising SEQ ID NO:34 has at least one of its internucleoside linkages which is modified, preferably as a PNms linkage, preferably at least two of them. In an embodiment, a PNms linkage is present in the central region of the oligonucleotide and not in its wings.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:34 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:34 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE. A most preferred oligonucleotide comprising SEQ ID NO:34 is represented by SEQ ID NO:35: eGseGseC*seAseAsdTsdUpNmsdCpNmsdTsdTsdTsdCsdTsdTsdGseAseAseAseAseT (SEQ ID NO:35)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON18) (SEQ ID NO: 36)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:36. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:36 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:36 present in the gap of said oligonucleotide (C at position 8 and at position 12 of SEQ ID NO:36) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:36 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:36 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:36 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NO:36 is fully modified with a PS backbone.
In an embodiment, the oligonucleotide comprising SEQ ID NO:36 has at least one of its internucleoside linkages which is modified, preferably as a PNms linkage, preferably at least two of them. In an embodiment, a PNms linkage is present in the central region of the oligonucleotide and not in its wings.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:36 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:36 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:36 is represented by SEQ ID NO:37: eGseGseC*seAseAsdTsdUsdCpNmsdTpNmsdTsdTsdCsdTsdTsdGseAseAseAseAseT (SEQ ID NO:37)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON19) (SEQ ID NO: 38) In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:38. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:38 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:38 present in the gap of said oligonucleotide (C at position 8 and at position 12 of SEQ ID NO:38) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:38 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:38 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:38 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO: 38 is fully modified with a PS backbone.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
In an embodiment, the oligonucleotide comprising SEQ ID NO:38 has at least one of its internucleoside linkages which is modified, preferably as a PNms linkage, preferably at least two of them. In an embodiment, a PNms linkage is present in the central region of the oligonucleotide and not in its wings.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:38 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:38 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:38 is represented by SEQ ID NO:39: eGseGoeC*oeAoeAsdTsdUsdCpNmsdTpNmsdTsdTsdCsdTsdTsdGseAoeAoeAoeAseT (SEQ ID NO:39)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAAATCTTTCTTGAAAAT (AON22) (SEQ ID NO: 40)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:40. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:40 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:40 present in the gap of said oligonucleotide (C at position 8 and at position 12 of SEQ ID NQ:40) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NQ:40 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:40 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NQ:40 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:40 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:40 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:40 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NQ:40 is represented by SEQ ID NO:41 : eGseGseC*seAseAsdAsdTsdCsdTsdTsdTsdCsdTsdTsdGseAseAseAseAseT (SEQ ID NO:41)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATTGTTTCTTGAAAAT (AON23) (SEQ ID NO: 42)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:42. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:42 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:42 present in the gap of said oligonucleotide (C at position 12 of SEQ ID NO:42) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:42 are 5- m ethyl cytosines. In an embodiment, the oligonucleotide comprising SEQ ID NO:42 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:42 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:42 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:42 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:42 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:42 is represented by SEQ ID NO:43: eGseGseC*seAseAsdTsdTsdGsdTsdTsdTsdCsdTsdTsdGseAseAseAseAseT (SEQ ID NO:43)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATTGATTCTTGAAAAT (AON24) (SEQ ID NO: 44)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:44. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:44 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:44 present in the gap of said oligonucleotide (C at position 12 of SEQ ID NO:44) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:44 are 5- methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:44 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:44 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:44 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:44 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:44 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:44 is represented by SEQ ID NO:45: eGseGseC*seAseAsdTsdTsdGsdAsdTsdTsdCsdTsdTsdGseAseAseAseAseT (SEQ ID NO:45)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON25) (SEQ ID NO: 46)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:46. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:46 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:46 present in the gap of said oligonucleotide (C at position 7 and C at position 1 1 of SEQ ID NO:46) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:46 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:46 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:46 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:46 is fully modified with a PS backbone.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate. In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:46 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:46 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:46 is represented by SEQ ID NO:47: eGseC*oeAoeAoeTsdUsdCsdTsdTsdTsdCsdTsdTsdGsdAseAoeAoeAoeTseG (SEQ ID NO:47)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATTCTAACTTGAAAAT (AON26) (SEQ ID NO: 48)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:48. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:48 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:48 present in the gap of said oligonucleotide (C at position 8 and at position 12 of SEQ ID NO:48) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:48 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:48 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:48 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:48 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:48 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:48 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:48 is represented by SEQ ID NO:49: eGseGseC*seAseAsdTsdTsdCsdTsdAsdAsdCsdTsdTsdGseAseAseAseAseT (SEQ ID NO:49)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATTCTTAGTTGAAAAT (AON27) (SEQ ID NO: 50)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:50. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NQ:50 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NQ:50 present in the gap of said oligonucleotide (C at position 8 of SEQ ID NO:50) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:50 are 5- m ethyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:50 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NQ:50 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:50 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:50 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:50 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NQ:50 is represented by SEQ ID NO:51 : eGseGseC*seAseAsdTsdTsdCsdTsdTsdAsdGsdTsdTsdGseAseAseAseAseT (SEQ ID NO:51)
In an embodiment the antisense oligonucleotide comprises the following base sequence: CAATTCTTTCTTGAAAAT (AON28) (SEQ ID NO: 52)
In this embodiment, the antisense oligonucleotide has a length of 18 to 50 nucleotides and comprises the base sequence SEQ ID NO:52. The length may be 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:52 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:52 present in the gap of said oligonucleotide (C at position 6 and at position 10 of SEQ ID NO:52) are not methylated. In another embodiment, and preferably all Cs of the oligonucleotide comprising SEQ ID NO:52 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:52 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:52 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:52 is fully modified with a PS backbone. In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:52 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:52 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 4 nucleotides. Most preferably both wings comprise 4 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:52 is represented by SEQ ID NO:53: eC*seAseAseTsdTsdCsdTsdTsdTsdCsdTsdTsdGsdAseAseAseAseT (SEQ ID NO:53)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATTCTTTCTTGAAAA (AON29) (SEQ ID NO: 54)
In this embodiment, the antisense oligonucleotide has a length of 18 to 50 nucleotides and comprises the base sequence SEQ ID NO:54. The length may be 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:54 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:54 present in the gap of said oligonucleotide (C at position 7 and at position 11 of SEQ ID NO:54) are not methylated. In another embodiment, and preferably all Cs of the oligonucleotide comprising SEQ ID NO:54 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:54 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:54 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:54 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:54 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:54 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 4 nucleotides. Most preferably both wings comprise 4 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:54 is represented by SEQ ID NO:55: eGseC*seAseAsdTsdTsdCsdTsdTsdTsdCsdTsdTsdGseAseAseAseA (SEQ ID NO:55) In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATTCTTTCTTGAAA (AON30) (SEQ ID NO: 56)
In this embodiment, the antisense oligonucleotide has a length of 18 to 50 nucleotides and comprises the base sequence SEQ ID NO:56. The length may be 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:56 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:56 present in the gap of said oligonucleotide (C at position 8 and at position 12 of SEQ ID NO:56) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:56 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:56 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:56 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:56 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:56 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:56 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise4 nucleotides. Most preferably both wings comprise 4 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:56 is represented by SEQ ID NO:57: eGseGseC*seAsdAsdTsdTsdCsdTsdTsdTsdCsdTsdTseGseAseAseA (SEQ ID NO:57)
In an embodiment the antisense oligonucleotide comprises the following base sequence: AATTCTTTCTTGAAAA (AON31) (SEQ ID NO: 58)
In this embodiment, the antisense oligonucleotide has a length of 16 to 50 nucleotides and comprises the base sequence SEQ ID NO:58. The length may be 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces. In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:58 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:58 present in the gap of said oligonucleotide (C at positions 5 and 9 of SEQ ID NO:58) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:58 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:58 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:58 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:58 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:58 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:58 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:58 is represented by SEQ ID NO:59: eAseAseTsdTsdCsdTsdTsdTsdCsdTsdTsdGsdAseAseAseA (SEQ ID NO:59)
In an embodiment the antisense oligonucleotide comprises the following base sequence: CAATTCTTTCTTGAAA (AON32) (SEQ ID NO: 60)
In this embodiment, the antisense oligonucleotide has a length of 16 to 50 nucleotides and comprises the base sequence SEQ ID NO:60. The length may be 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NQ:60 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NQ:60 present in the gap of said oligonucleotide (C at position 6 and position 10 of SEQ ID NQ:60) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NQ:60 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:60 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NQ:60 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:60 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:60 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:60 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 3 nucleotides. Most preferably both wings comprises nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NQ:60 is represented by SEQ ID NO:61 : eC*seAseAsdTsdTsdCsdTsdTsdTsdCsdTsdTsdGseAseAseA (SEQ ID NO:61)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATTCTTTCTTGAA (AON33) (SEQ ID NO: 62)
In this embodiment, the antisense oligonucleotide has a length of 16 to 50 nucleotides and comprises the base sequence SEQ ID NO:62. The length may be 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:62 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:62 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO:62) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:62 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:62 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:62 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, , the oligonucleotide comprising SEQ ID NO:62 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:62 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:62 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 3 nucleotides. Most preferably both wings comprise 3 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:62 is represented by SEQ ID NO:63: eGseC eAsdAsdTsdTsdCsdTsdTsdTsdCsdTsdTseGseAseA (SEQ ID NO:63)
In an embodiment the antisense oligonucleotide comprises the following base sequence: ATTCTTTCTTGAAAATGGCA (AON34) (SEQ ID NO: 64)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:64. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:64 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:64 present in the gap of said oligonucleotide (C at position 8 of SEQ ID NO:64) are not methylated. In another embodiment, and preferably all Cs of the oligonucleotide comprising SEQ ID NO:64 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:64 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:64 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:64 is fully modified with a PS backbone.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:64 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:64 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:64 is represented by SEQ ID NO:65: eAseToeToeC*oeTsdTsdTsdCsdTsdTsdGsdAsdAsdAsdAseToeGoeGoeC*seA (SEQ ID NO:65)
In an embodiment the antisense oligonucleotide comprises the following base sequence: ATTCTTTCTTGAAAATGGCA (AON35) (SEQ ID NO: 66)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:66. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:66 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:66 present in the gap of said oligonucleotide (C at position 8 of SEQ ID NO:66) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:66 are 5- m ethyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:66 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:66 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:66 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:66 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:66 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:66 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:66 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:66 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has two distinct internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
A most preferred oligonucleotide comprising SEQ ID NO:66 is represented by SEQ ID NO:67: eAseT PNdmieT PNdmieC*PNdmieTsdTsdTsdCsdTsdTsdGsdAsdAsdAsdAseT PNdmieGpNdmieGpNdmieC*seA (SEQ ID NO:67)
In an embodiment the antisense oligonucleotide comprises the following base sequence:
GGCAATTCTTTCTTGAAAAT (AON36) (SEQ ID NO: 68) In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:68. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:68 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:68 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:68) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:68 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:68 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:68 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:68 is fully modified with a PS backbone.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:68 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:68 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:68 is represented by SEQ ID NO:69: eGseGoeC*oeAoeAsdTsdTsdCsdTsdTsdTsdCsdTsdTsdGseAoeAoeAoeAseT (SEQ ID NO:69)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATTCTTTCTTGAAAAT (AON37) (SEQ ID NO: 70)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:70. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces. In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:70 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:70 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NQ:70) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NQ:70 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:70 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NQ:70 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:70 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NQ:70 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NQ:70 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NQ:70 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has two distinct internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:70 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:70 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NQ:70 is represented by SEQ ID NO:71 : eGseGpNdmieC*PNdmieApNdmieAsdT sdT sdCsdT sdT sdT sdCsdT sdTsdGseApNdmieApNdmieApNdmieAseT (SEQ ID NO:71)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATTCTTTCTTGAAAAT (AON38) (SEQ ID NO: 72)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:72. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:72 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:72 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:72) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:72 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:72 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:72 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:72 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:72 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:72 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
A most preferred oligonucleotide comprising SEQ ID NO:72 is represented by SEQ ID NO:73: eGseGoeC*seAoeAsdTsdTsdCsdTsdTsdTsdCsdTsdTsdGseAoeAseAoeAseT (SEQ ID NO:73)
In an embodiment the antisense oligonucleotide comprises the following base sequence: AGCTGGCAATTCTTTCTTGA (AON39) (SEQ ID NO: 74)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:74. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:74 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:74 present in the gap of said oligonucleotide (C at position 7 and position 12 of SEQ ID NO:74) are not methylated. In another embodiment, and preferably all Cs of the oligonucleotide comprising SEQ ID NO:74 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:74 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:74 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:74 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:74 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:74 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
A most preferred oligonucleotide comprising SEQ ID NO:74 is represented by SEQ ID NO:75: eAseGoeC*oeToeGsdGsdCsdAsdAsdTsdTsdCsdTsdTsdTseC*oeToeToeGseA (SEQ ID NO:75)
In an embodiment the antisense oligonucleotide comprises the following base sequence: AGCTGGCAATTCTTTCTTGA (AQN40) (SEQ ID NO: 76)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:76. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:76 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:76 present in the gap of said oligonucleotide (C at position 7 and position 12 of SEQ ID NO:76) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:76 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:76 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:76 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:76 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:76 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:76 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:76 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has two distinct internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:76 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:76 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:76 is represented by SEQ ID NO:77: eAseGpNdmieC*PNdmieT PNdmieGsdGsdCsdAsdAsdTsdTsdCsdTsdTsdTseC*PNdmieT PNdmieT PNdmieGseA (SEQ ID NO:77)
In an embodiment the antisense oligonucleotide comprises the following base sequence: AGCTGGCAATTCTTTCTTGA (AON41) (SEQ ID NO: 78)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:78. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:78 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:78 present in the gap of said oligonucleotide (C at position 7 and position 12 of SEQ ID NO:78) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:78 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:78 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:78 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:78 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:78 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:78 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
A most preferred oligonucleotide comprising SEQ ID NO:78 is represented by SEQ ID NO:79: eAseGoeC*seToeGsdGsdCsdAsdAsdTsdTsdCsdTsdTsdTseC*oeTseToeGseA (SEQ ID NO:79)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GAAGAGCTGGCAATTCTTTC (AON42) (SEQ ID NO: 80)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NQ:80. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NQ:80 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NQ:80 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NQ:80) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NQ:80 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:80 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NQ:80 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:80 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:80 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:80 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
A most preferred oligonucleotide comprising SEQ ID NO:80 is represented by SEQ ID NO:81 : eGseAoeAoeGoeAsdGsdCsdTsdGsdGsdCsdAsdAsdTsdTseC*oeToeToeTseC* (SEQ ID NO:81)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GAAGAGCTGGCAATTCTTTC (AON43) (SEQ ID NO: 82)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:82. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:82 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:82 present in the gap of said oligonucleotide (C at position 7 and position 1 1 of SEQ ID NO:82) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:82 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:82 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:82 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:82 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:82 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:82 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:82 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has two distinct internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate. In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:82 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:82 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:82 is represented by SEQ ID NO:83: eGseApNdmieApNdmieGpNdmieAsdGsdCsdT sdGsdGsdCsdAsdAsdT sdT seC*PNdmieT PNdmieT PNdmieT seC* (SEQ ID NO:83)
In an embodiment the antisense oligonucleotide comprises the following base sequence: TGGCAATUCTTTCTTGAAAA (AON44) (SEQ ID NO: 84)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:84. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:84 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:84 present in the gap of said oligonucleotide (C at position 9 and position 13 of SEQ ID NO:84) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:84 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:84 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:84 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:84 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:84 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:84 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE. In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
A most preferred oligonucleotide comprising SEQ ID NO:84 is represented by SEQ ID NO:85: eTseGoeGoeC*oeAsdAsdTsdUsdCsdTsdTsdTsdCsdTsdTseGoeAoeAoeAseA (SEQ ID NO:85)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON45) (SEQ ID NO: 86)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:86. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:86 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:86 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO:86) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:86 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:86 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:86 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:86 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:86 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:86 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:86 comprises two distinct modified internucleoside linkages, preferably dmi-Phosphoramidate and phosphorothioate.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi- Phosphoramidate and phosphorothioate. In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:86 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:86 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:86 is represented by SEQ ID NO:87: eGseC*PNdmieAoeApNdmieT sdUsdCsdT sdTsdTsdCsdTsdTsdGsdAseAoeAoeAoeT PNdmieG (SEQ I D
NO:87)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON46) (SEQ ID NO: 88)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:88. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:88 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:88 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:88) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:88 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:88 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:88 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:88 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:88 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:88 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:88 comprises two distinct modified internucleoside linkages, preferably dmi-Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide hasthree distinct internucleoside linkages, preferably phosphodiester, dmi-Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:88 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:88 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:88 is represented by SEQ ID NO:89: eGseGpNdmieC*oeApNdmieAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdGseAoeAoeAoeApNdmieT (SEQ ID NO:89)
In an embodiment the antisense oligonucleotide comprises the following base sequence: TGGCAATUCTTTCTTGAAAA (AON47) (SEQ ID NO: 90)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:90. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NQ:90 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NQ:90 present in the gap of said oligonucleotide (C at position 9 and position 13 of SEQ ID NQ:90) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NQ:90 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:90 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NQ:90 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:90 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NQ:90 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:90 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:90 comprises two distinct modified internucleoside linkages, preferably dmi-Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi-Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:90 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:90 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NQ:90 is represented by SEQ ID NO:91 : eTseGpNdmieGoeC*PNdmieAsdAsdTsdUsdCsdTsdTsdTsdCsdTsdTseGoeAoeAoeApNdmieA (SEQ ID NO:91)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAA (AON48) (SEQ ID NO: 92)
In this embodiment, the antisense oligonucleotide has a length of 18 to 50 nucleotides and comprises the base sequence SEQ ID NO:92. The length may be 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:92 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:92 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:92) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:92 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:92 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:92 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:92 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:92 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:92 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 4 nucleotides. Most preferably both wings comprise 4 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:92 is represented by SEQ ID NO:93: eGseGseC*seAsdAsdTsdUsdCsdTsdTsdTsdCsdTsdTseGseAseAseA (SEQ ID NO:93)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAA (AON49) (SEQ ID NO: 94)
In this embodiment, the antisense oligonucleotide has a length of 18 to 50 nucleotides and comprises the base sequence SEQ ID NO:94. The length may be 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:94 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:94 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:94) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:94 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:94 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:94 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, = the oligonucleotide comprising SEQ ID NO:94 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:94 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:94 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 1 or 2 or 3, most preferably 1.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:94 comprises two distinct modified internucleoside linkages, preferably dmi-Phosphoramidate and phosphorothioate. In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi- Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:94 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:94 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 4 nucleotides. Most preferably both wings comprise 4 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:94 is represented by SEQ ID NO:95: eGseGpNdmieC*oeAsdAsdTsdUsdCsdTsdTsdTsdCsdTsdTseGoeAoeApNdmieA (SEQ ID NO:95)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AQN50) (SEQ ID NO: 96)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:96. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:96 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:96 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO:96) are not methylated. In another embodiment, and preferably all Cs of the oligonucleotide comprising SEQ ID NO:96 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:96 has at least one of its internucleoside linkages which is modified, preferably as a PNms linkage, preferably at least two of them. In an embodiment, a PNms linkage is present in the central region of the oligonucleotide and not in its wings. In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl-phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:96 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:96 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:96 is represented by SEQ ID NO:97: eGseC*oeAoeAoeTsdUpNmsdCpNmsdTsdTsdTsdCsdTsdTsdGsdAseAoeAoeAoeTseG (SEQ ID NO:97)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON51) (SEQ ID NO: 98)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:98. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:98 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:98 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO:98) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:98 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:98 has at least one of its internucleoside linkages which is modified, preferably as a PNms linkage, preferably at least two of them. In an embodiment, a PNms linkage is present in the central region of the oligonucleotide and not in its wings.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:98 comprises one modified internucleoside linkage, preferably phosphorothioate.
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl-phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:98 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:98 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE. A most preferred oligonucleotide comprising SEQ ID NO:98 is represented by SEQ ID NO:99: eGseC*oeAoeAoeTsdUsdCpNmsdTpNmsdTsdTsdCsdTsdTsdGsdAseAoeAoeAoeTseG (SEQ ID NO:99)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON52) (SEQ ID NO: 100)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:100. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NQ:100 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NQ:100 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NQ:100) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NQ:100 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:100 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 100 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:100 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings or in the central part of the oligonucleotide comprising SEQ ID NQ:100 is a mesyl- phosphoramidate (PNms) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NQ:100 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2. Preferably, the central part of the oligonucleotide comprising SEQ ID NQ:100 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NQ:100 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
In an embodiment, 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NQ:100 and no PNms linkage is present in the wings of said oligonucleotide. In this embodiment, the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl-phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID N0:100 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID N0:100 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NQ:100 is represented by SEQ ID NQ:101 : eGseC*PNmseApNmseAoeT sd UsdCpNmsdT PNmsdT sdT sdCsdTsdT sdGsd AseAoeAoeAoeT seG (SEQ I D NQ:101)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON53) (SEQ ID NO: 102)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:102. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NQ:102 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NQ:102 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NQ:102) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NQ:102 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:102 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 102 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, , the oligonucleotide comprising SEQ ID NQ:102 is fully modified with a PS backbone.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:102 has at least one of its internucleoside linkages which is modified, preferably as a PNms linkage, preferably at least two of them. In an embodiment, a PNms linkage is present in the central region of the oligonucleotide and not in one wing (or in both wings).
In an embodiment, the oligonucleotide comprising SEQ ID NQ:102 has at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl-phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:102 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:102 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NQ:102 is represented by SEQ ID NQ:103: eGseC*oeAoeAoeTsdUsdCpNmsdTpNmsdTsdTsdCsdTsdTsdGsdAseAseAseAseTseG (SEQ ID NQ:103)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON54) (SEQ ID NO: 104)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:104. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NQ:104 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NQ:104 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO: 104) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NQ:104 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:104 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 104 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:104 is fully modified with a PS backbone.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:104 has at least one of its internucleoside linkages which is modified, preferably as a PNms linkage, preferably at least 2, 3, 4, 5, of them. In an embodiment, a PNms linkage is present in the central region of the oligonucleotide and not in its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:104 has at least one internucleoside linkages in one wing (or in both wings) which is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, the oligonucleotide has distinct internucleoside linkages, preferably phosphodiester, mesyl-phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:104 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:104 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NQ:104 is represented by SEQ ID NQ:105: eGseC*seAseAseTsdUsdCpNmsdTpNmsdTsdTsdCsdTsdTsdGsdAseAoeAoeAoeTseG (SEQ ID NQ:105)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON55) (SEQ ID NO: 106)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:106. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NQ:106 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NQ:106 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NQ:106) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NQ:106 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:106 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 106 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, , the oligonucleotide comprising SEQ ID NQ:106 is fully modified with a PS backbone.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:106 has at least one of its internucleoside linkages which is modified, preferably as a PNms linkage, preferably at least 2, 3, 4, 5 of them. In an embodiment, a PNms linkage is present in the central region of the oligonucleotide and not in one of its wings (or not in both wings).
In an embodiment, the oligonucleotide comprising SEQ ID NO:106 has at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl-phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:106 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:106 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NQ:106 is represented by SEQ ID NQ:107: eGseGoeC*oeAoeAsdTsdUpNmsdCpNmsdTsdTsdTsdCsdTsdTsdGseAoeAoeAoeAseT (SEQ ID NQ:107)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON56) (SEQ ID NO: 108)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:108. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NQ:108 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NQ:108 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO: 108) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NQ:108 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:108 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 108 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:108 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings or in the central part of the oligonucleotide comprising SEQ ID NQ:108 is a mesyl- phosphoramidate (PNms) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:108 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2. Preferably, the central part of the oligonucleotide comprising SEQ ID NO:108 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2. In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NQ:108 comprises two distinct modified internucleoside linkages, preferably mesyl-phosphoramidate and phosphorothioate.
In an embodiment, 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NQ:108 and no PNms linkage is present in the wings of said oligonucleotide. In this embodiment, the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:108 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:108 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NQ:108 is represented by SEQ ID NQ:109: eGseGpNmseC*PNmseAoeAsdT sdUsdCpNmsdT PNmsdT sdT sdCsdT sdT sdGseAoeAoeAoeAseT (SEQ ID NQ:109)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON57) (SEQ ID NO: 110)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:110. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NQ:110 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NQ:110 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NQ:110) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:110 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:110 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:110 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:110 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NQ:110 is a mesyl-phosphoramidate (PNms) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:1 10 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NQ:1 10 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
In an embodiment, 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NQ:110 and no PNms linkage is present in the wings of said oligonucleotide. In this embodiment, the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester .mesyl-phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:110 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:110 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:110 is represented by SEQ ID NO:1 11 : eGseGpNmseC*PNmseAoeAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdGseAoeAoeAoeAseT (SEQ ID NO:111)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON58) (SEQ ID NO: 112)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:112. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:112 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:112 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO: 112) are not methylated. In another embodiment, and preferably all Cs of the oligonucleotide comprising SEQ ID NO:112 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:112 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:112 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:112 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:112 is a mesyl-phosphoramidate (PNms) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:1 12 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:1 12 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
In an embodiment, 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:112 and no PNms linkage is present in the wings of said oligonucleotide. In this embodiment, the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:112 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:112 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:112 is represented by SEQ ID NO:1 13: eGseGoeC*PNmseApNmseAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdGseAoeAoeAoeAseT (SEO ID NO:113)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON59) (SEQ ID NO: 114)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:114. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:114 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:114 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO: 114) are not methylated. In another embodiment, and preferably all Cs of the oligonucleotide comprising SEQ ID NO:114 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:114 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:114 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, , the oligonucleotide comprising SEQ ID NO:1 14 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:114 is a mesyl-phosphoramidate (PNms) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:114 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:114 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
In an embodiment, 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:114 and no PNms linkage is present in the wings of said oligonucleotide. In this embodiment, the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate. In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl-phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:114 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:114 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:114 is represented by SEQ ID NO:1 15: eGseGoeC*oeAoeAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdGseAoeAoeApNmseApNmseT (SEQ ID NO:1 15)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AQN60) (SEQ ID NO: 116)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:116. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:116 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:116 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO: 116) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:116 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:116 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:116 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:116 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:116 is a mesyl-phosphoramidate (PNms) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:1 16 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2. In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:1 16 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
In an embodiment, 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:116 and no PNms linkage is present in the wings of said oligonucleotide. In this embodiment, the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:116 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:116 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:116 is represented by SEQ ID NO:117: eGseGoeC*oeAoeAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdGseAoeApNmseApNmseAoeT (SEQ ID NO:117).
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON61) (SEQ ID NO: 121)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:121 . The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:121 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:121 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO: 121) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:121 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:121 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:121 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:121 is fully modified with a PS backbone.
In an embodiment, in the gap of the oligonucleotide comprising SEQ ID NO:121 , at least one sugar modification is present, preferably at least one 2-Omethyl sugar modification is present in the gap of said oligonucleotide. Most preferably, at position 7 of SEQ ID NO: 121 , a 2-Omethyl sugar modification is present.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:121 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:121 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:121 is represented by SEQ ID NO:122: eGseC*oeAoeAoeTsdUsmCsdTsdTsdTsdCsdTsdTsdGsdAseAoeAoeAoeTseG (SEQ ID NO:122)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON62) (SEQ ID NO: 123)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:123. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:123 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:123 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:123) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:123 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:123 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 123 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:123 is fully modified with a PS backbone. In an embodiment, in the gap of the oligonucleotide comprising SEQ ID NO:123, at least one sugar modification is present, preferably at least one 2-Omethyl sugar modification is present in the gap of said oligonucleotide. Most preferably, at position 7 of SEQ ID NO: 123, a 2-Omethyl sugar modification is present.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:123 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:123 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:123 is represented by SEQ ID NO:124: eGseGoeC*oeAoeAsdTsmUsdCsdTsdTsdTsdCsdTsdTsdGseAoeAoeAoeAseT (SEQ ID NO:124)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON63) (SEQ ID NO: 125)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:125. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:125 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:125 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO:125) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:125 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:125 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:125 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:125 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:125 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO: 125 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 1 or 2 or 3, most preferably 1
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO: 125 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi- Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:125 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:125 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:125 is represented by SEQ ID NO:126: eGseC*oeAoeAoeTsdUsdCsdTsdTsdTsdCsdTsdTsdGsdAseAoeAoeAoeTpNdmieG (SEQ ID NO:126)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON64) (SEQ ID NO: 127)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:127. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:127 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:127) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:127 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:127 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 127 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:127 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:127 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:127 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 1 or 2 or 3, most preferably 1 .
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:127 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi- Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:127 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:127 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:127 is represented by SEQ ID NO:128: eGseGoeC*oeAoeAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdGseAoeAoeAoeApNdmieT (SEQ ID NO:128)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON65) (SEQ ID NO: 129)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:129. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:129 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO:129) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:129 are 5- methyl cytosines. In an embodiment, the oligonucleotide comprising SEQ ID NO:129 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 129 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:129 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:129 is a mesyl-phosphoramidate (PNms) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:129 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO: 129 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
In an embodiment, 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:129 and no PNms linkage is present in the wings of said oligonucleotide. In this embodiment, the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
In an embodiment, in the gap of the oligonucleotide comprising SEQ ID NO:129, at least one sugar modification is present, preferably at least one 2-Omethyl sugar modification is present in the gap of said oligonucleotide. Most preferably, at position 7 of SEQ ID NO: 129, a 2-Omethyl sugar modification is present.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:129 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:129 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:129 is represented by SEQ ID NQ:130: eGseC*oeAoeAoeTsdUpNmsmCpNmsdTsdTsdTsdCsdTsdTsdGsdAseAoeAoeAoeTseG (SEQ ID NQ:130) In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON66) (SEQ ID NO: 131)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:131 . The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:131 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO:131) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:131 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:131 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 131 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:131 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:131 is a mesyl-phosphoramidate (PNms) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:131 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:131 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
In an embodiment, 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:131 and no PNms linkage is present in the wings of said oligonucleotide. In this embodiment, the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate. In an embodiment, in the gap of the oligonucleotide comprising SEQ ID NO:131 , at least one sugar modification is present, preferably at least one 2-Omethyl sugar modification is present in the gap of said oligonucleotide. Most preferably, at position 7 of SEQ ID NO: 131 , a 2-Omethyl sugar modification is present.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:131 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:131 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:131 is represented by SEQ ID NO:132: eGseC*oeAoeAoeTsdUsmCpNmsdTpNmsdTsdTsdCsdTsdTsdGsdAseAoeAoeAoeTseG (SEQ ID NO:132)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON67) (SEQ ID NO: 133)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:133. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:133 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:133 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:133) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:133 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:133 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:133 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:133 is a mesyl-phosphoramidate (PNms) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:133 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2. In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:133 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
In an embodiment, 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:133 and no PNms linkage is present in the wings of said oligonucleotide. In this embodiment, the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:133 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:133 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:133 is represented by SEQ ID NO:134: eGseGoeC*oeAoeAsdTsmUpNmsdCpNmsdTsdTsdTsdCsdTsdTsdGseAoeAoeAoeAseT (SEQ ID NO:134)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON68) (SEQ ID NO: 135)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:135. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:135 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:135 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:135) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:135 are 5- methyl cytosines. In an embodiment, the oligonucleotide comprising SEQ ID NO:135 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 135 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:135 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:135 is a mesyl-phosphoramidate (PNms) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:135 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:135 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
In an embodiment, 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:135 and no PNms linkage is present in the wings of said oligonucleotide. In this embodiment, the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:135 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:135 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
In an embodiment, in the gap of the oligonucleotide comprising SEQ ID NO:135, at least one sugar modification is present, preferably at least one 2-Omethyl sugar modification is present in the gap of said oligonucleotide. Most preferably, at position 7 of SEQ ID NO: 135, a 2-Omethyl sugar modification is present.
A most preferred oligonucleotide comprising SEQ ID NO:135 is represented by SEQ ID NO:136: eGseGoeC*oeAoeAsdTsmUsdCpNmsdTpNmsdTsdTsdCsdTsdTsdGseAoeAoeAoeAseT (SEQ ID NO:136)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON69) (SEQ ID NO: 137)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:137. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:137 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:137 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO: 137) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:137 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:137 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 137 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:137 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:137 is a mesyl-phosphoramidate (PNms) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO: 137 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO: 137 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
In an embodiment, 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:137 and no PNms linkage is present in the wings of said oligonucleotide. In this embodiment, the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:137 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:137 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 1 or 2 or 3, most preferably 1 .
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:137 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:137 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:137 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:137 is represented by SEQ ID NO:138: eGseC*oeAoeAoeT sdUpNmsdCpNmsdT sdT sdTsdCsdTsdTsdGsdAseAoeAoeAoeT PNdmieG (SEQ I D NO:138)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AQN70) (SEQ ID NO: 139)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:139. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:139 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:139 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO:139) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:139 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:139 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 139 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:139 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:139 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:139 is a mesyl-phosphoramidate (PNms) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:139 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:139 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
In an embodiment, 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:139 and no PNms linkage is present in the wings of said oligonucleotide. In this embodiment, the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:139 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:139 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 1 or 2 or 3, most preferably 1 .
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:139 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:139 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:139 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:139 is represented by SEQ ID NO:140: eGseC*oeAoeAoeT sdUsdCpNmsdT PNmsdT sdT sdCsdTsdTsdGsdAseAoeAoeAoeT PNdmieG (SEO I D NO:140)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON71) (SEQ ID NO: 141)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:141 . The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:141 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:141 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:141) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:141 are 5- methyl cytosines.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:141 is a mesyl-phosphoramidate (PNms) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:141 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:141 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
In an embodiment, 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:141 and no PNms linkage is present in the wings of said oligonucleotide. In this embodiment, the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate. In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:141 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:141 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 1 or 2 or 3, most preferably 1 .
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:141 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide comprising SEQ ID NO:141 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 141 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:141 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:141 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:141 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:141 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:141 is represented by SEQ ID NO:142: eGseGoeC*oeAoeAsdT sdUpNmsdCpNmsdT sdT sdTsdCsdTsdT sdGseAoeAoeAoeApNdmieT (SEQ ID NO:142)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON72) (SEQ ID NO: 143)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:143. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:143 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:143 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:143) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:143 are 5- methyl cytosines.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:143 is a mesyl-phosphoramidate (PNms) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:143 comprises 1 or 2 or 3 or 4 or 5 PNms linkages, more preferably 2 or 3 or 4, most preferably 2.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:143 comprises two distinct modified internucleoside linkages, preferably mesyl- phosphoramidate and phosphorothioate.
In an embodiment, 1 , 2, 3, 4, 5 PNms linkages are present in the central part of the oligonucleotide comprising SEQ ID NO:143 and no PNms linkage is present in the wings of said oligonucleotide. In this embodiment, the internucleoside linkage in the wings of said oligonucleotide are chosen from phophodiesters linkage, mesyl-phosphoramidate and phosphorothioate.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:143 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:143 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 1 or 2 or 3, most preferably 1 .
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:143 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioateln an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, mesyl- phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide comprising SEQ ID NO:143 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 143 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:143 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:143 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:143 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:143 is represented by SEQ ID NO:144: eGseGoeC*oeAoeAsdT sdUsdCpNmsdT PNmsdT sdT sdCsdTsdT sdGseAoeAoeAoeApNdmieT (SEQ ID NO:144)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GTAAATCATTCCCACCACAC (AON73) (SEQ ID NO: 173)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:173. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:173 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:173 present in the gap of said oligonucleotide (C at positions 7, 11 , 12, 13, 15 of SEQ ID NO:173) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:173 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:173 has at least one of its internucleoside linkages which is modified, preferably as a phosphoroth ioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:173 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:173 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:173 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:173 is represented by SEQ ID NO:174: eGseTseAseAseAsdTsdCsdAsdTsdTsdCsdCsdCsdAsdCseC*seAseC*seAseC* (SEQ ID NO:174).
In an embodiment the antisense oligonucleotide comprises the following base sequence:
ATGTAAATCATTCCCACCAC (AON74) (SEQ ID NO: 175) In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:175. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:175 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:175 present in the gap of said oligonucleotide (C at positions 9, 13-15 of SEQ ID NO: 175) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:175 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:175 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:175 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:175 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:175 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:175 is represented by SEQ ID NO:176: eAseTseGseTseAsdAsdAsdTsdCsdAsdTsdTsdCsdCsdCseAseC*seC*seAseC* (SEQ ID NO:176).
In an embodiment the antisense oligonucleotide comprises the following base sequence: GAATGTAAATCATTCCCACC (AON75) (SEQ ID NO: 177)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:177. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:177 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:177 present in the gap of said oligonucleotide (C at position 11 and position 15 of SEQ ID NO:177) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO: 177 are 5-methylcytosines. In an embodiment, the oligonucleotide comprising SEQ ID NO:177 has at least one of its internucleoside linkages which is modified, preferably as a phosphoroth ioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:177 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:177 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:177 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:177 is represented by SEQ ID NO:178: eGseAseAseTseGsdTsdAsdAsdAsdTsdCsdAsdTsdTsdCseC*seC*seAseC*seC* (SEQ ID NO:178)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GTGAATGTAAATCATTCCCA (AON76) (SEQ ID NO: 179).
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:179. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:179 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:179present in the gap of said oligonucleotide (C at position 13 of SEQ ID NO:179) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:179 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:179 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:179 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:179 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:179 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:179 is represented by SEQ ID NQ:180: eGseTseGseAseAsdTsdGsdTsdAsdAsdAsdTsdCsdAsdTseTseC*seC*seC*seA (SEQ ID NO:180)
In an embodiment the antisense oligonucleotide comprises the following base sequence: CAGTGAATGTAAATCATTCC (AON77) (SEQ ID NO: 181).
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:181 . The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:181 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:181 present in the gap of said oligonucleotide (C at position 15 of SEQ ID NO:181) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:181 are 5- m ethyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:181 has at least one of its internucleoside linkages which is modified, preferably as a phosphoroth ioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO: 181 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:181 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:181 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:181 is represented by SEQ ID NO:182: eC*seAseGseTseGsdAsdAsdTsdGsdTsdAsdAsdAsdTsdCseAseTseTseC*seC* (SEQ ID NO:182)
In an embodiment the antisense oligonucleotide comprises the following base sequence: TCCAGTGAATGTAAATCATT (AON78) (SEQ ID NO: 183).
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:183. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces. In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:183 is 5- methylcytosine. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:183 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:183 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:183 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:183 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:183 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:183 is represented by SEQ ID NO:184: eTseC*seC*seAseGsdTsdGsdAsdAsdTsdGsdTsdAsdAsdAseTseC*seAseTseT (SEQ ID NO:184)
In an embodiment the antisense oligonucleotide comprises the following base sequence: TTTCCAGTGAATGTAAATCA (AON79) (SEQ ID NO: 185).
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:185. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:185 is 5- methylcytosine. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:185 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:185 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:185 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:185 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:185 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE. A most preferred oligonucleotide comprising SEQ ID NO:185 is represented by SEQ ID NO:186: eTseTseTseC*seC*sdAsdGsdTsdGsdAsdAsdTsdGsdTsdAseAseAseTseC*seA (SEQ ID NO:186)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GUAAATCATTCCCACCACAC (AON 80) (SEQ ID NO: 187).
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:187. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:187 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:187 present in the gap of said oligonucleotide (C at positions 7, 11-13, and 15 of SEQ ID NO:187) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:187 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:187 has at least one of its internucleoside linkages which is modified, preferably as a phosphoroth ioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:187 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:187 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:187 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:187 is represented by SEQ ID NO:188: eGseUseAseAseAsdTsdCsdAsdTsdTsdCsdCsdCsdAsdCseC*seAseC*seAseC* (SEQ ID NO:188)
In an embodiment the antisense oligonucleotide comprises the following base sequence: ATGUAAATCATTCCCACCAC (AON 81 (SEQ ID NO: 189).
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:189. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces. In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:189 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:189 present in the gap of said oligonucleotide (C at positions 9, 13-15, of SEQ ID NO:189) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:189 are 5- methyl cytosines.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:189 comprises one distinct modified internucleoside linkages, preferably phosphorothioate.
In an embodiment, the oligonucleotide comprising SEQ ID NO:189 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:189 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:189 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:189 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:189 is represented by SEQ ID NQ:190: eAseTseGseUseAsdAsdAsdTsdCsdAsdTsdTsdCsdCsdCseAseC*seC*seAseC* (SEQ ID NQ:190)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GAATGUAAATCATTCCCACC (AON 82 (SEQ ID NO: 191).
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:191 . The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:191 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:191 present in the gap of said oligonucleotide (C at position 11 and at position 15 of SEQ ID NO:191) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:191 are 5-methylcytosines.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:191 comprises one distinct modified internucleoside linkages, preferably phosphorothioate.
In an embodiment, the oligonucleotide comprising SEQ ID NO:191 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:191 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:191 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:191 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:191 is represented by SEQ ID NO:192: eGseAseAseTseGsdUsdAsdAsdAsdTsdCsdAsdTsdTsdCseC*seC*seAseC*seC* (SEQ ID NO:192)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GTGAATGUAAATCATTCCCA (AON 83 (SEQ ID NO: 193).
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:193. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:193 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:193 present in the gap of said oligonucleotide (C at position 13 of SEQ ID NO:193) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:193 are 5- m ethyl cytosines.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:193 comprises one distinct modified internucleoside linkages, preferably phosphorothioate.
In an embodiment, the oligonucleotide comprising SEQ ID NO:193 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:193 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:193 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:193 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:193 is represented by SEQ ID NO:194: eGseTseGseAseAsdTsdGsdUsdAsdAsdAsdTsdCsdAsdTseTseC*seC*seC*seA (SEQ ID NO:194)
In an embodiment the antisense oligonucleotide comprises the following base sequence: CAGTGAATGUAAATCATTCC (AON 84 (SEQ ID NO: 195).
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:195. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:195 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:195 present in the gap of said oligonucleotide (C at position 15 of SEQ ID NO:195) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:195 are 5- m ethyl cytosines.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:195 comprises one distinct modified internucleoside linkages, preferably phosphorothioate.
In an embodiment, the oligonucleotide comprising SEQ ID NO:195 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:195 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:195 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:195 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:195 is represented by SEQ ID NO:196: eC*seAseGseTseGsdAsdAsdTsdGsdUsdAsdAsdAsdTsdCseAseTseTseC*seC* (SEQ ID NO:196)
In an embodiment the antisense oligonucleotide comprises the following base sequence: TCCAGTGAATGUAAATCATT (AON 85 (SEQ ID NO: 197).
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:197. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:197 is 5- methylcytosine. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:197 are 5- methyl cytosines.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:197 comprises one distinct modified internucleoside linkages, preferably phosphorothioate.
In an embodiment, the oligonucleotide comprising SEQ ID NO:197 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:197 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:197 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:197 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:197 is represented by SEQ ID NO:198: eTseC*seC*seAseGsdTsdGsdAsdAsdTsdGsdUsdAsdAsdAseTseC*seAseTseT (SEQ ID NO:198)
In an embodiment the antisense oligonucleotide comprises the following base sequence: TTTCCAGTGAATGUAAATCA (AON 86 (SEQ ID NO: 199).
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:199. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:199 is 5- methylcytosine. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:199 are 5- methyl cytosines.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:199 comprises one distinct modified internucleoside linkages, preferably phosphorothioate.
In an embodiment, the oligonucleotide comprising SEQ ID NO:199 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:199 is fully modified with a PS backbone. In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:199 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:199 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:199 is represented by SEQ ID NQ:200: eTseTseTseC*seC*sdAsdGsdTsdGsdAsdAsdTsdGsdUsdAseAseAseTseC*seA (SEQ ID NQ:200)
In an embodiment the antisense oligonucleotide comprises the following base sequence: AGCGCCGGCAATTCTTTCTTG (AON 88 (SEQ ID NO: 203).
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:203. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NQ:203 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NQ:203 present in the gap of said oligonucleotide (C at positions 6, 9, 14 of SEQ ID NQ:203) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NQ:203 are 5- methyl cytosines.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NQ:203 comprises one distinct modified internucleoside linkages, preferably phosphorothioate.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:203 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NQ:203 is fully modified with a PS backbone.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:203 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:203 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NQ:203 is represented by SEQ ID NQ:204: eAseGoeC*oeGoeC*sdCsdGsdGsdCsdAsdAsdTsdTsdCsdTsdTseToeC*oeToeTseG (SEQ ID NQ:204) In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON89) (SEQ ID NO: 218)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:218. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:218 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:218 present in the gap of said oligonucleotide (C at position 7 and at position 11 of SEQ ID NO:218) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:218 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:218 has at least one of its internucleoside linkages which is modified, preferably as a phosphoroth ioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:218 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NO:218 is fully modified with a PS backbone.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate. In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:218 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:218 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:218 is represented by SEQ ID NO:219: eGseC*oeAseAoeTsdUsdCsdTsdTsdTsdCsdTsdTsdGsdAseAoeAseAoeTseG (SEQ ID NO:219)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AQN90) (SEQ ID NO: 220)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:220. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:220 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:220 present in the gap of said oligonucleotide (C at position 8 and at position 12 of SEQ ID NQ:220) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NQ:220 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:220 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NQ:220 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NQ:220 is fully modified with a PS backbone.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate. In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:220 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:220 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NQ:220 is represented by SEQ ID NO:221 : eGseGoeC*seAoeAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdGseAoeAseAoeAseT (SEQ ID NO:221)
In an embodiment the antisense oligonucleotide comprises the following base sequence: TGGCAATUCTTTCTTGAAAA (AON91) (SEQ ID NO: 222)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:222. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:222 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:222 present in the gap of said oligonucleotide (C at position 9 and at position 13 of SEQ ID NO:222) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:222 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:222 has at least one of its internucleoside linkages which is modified, preferably as a phosphoroth ioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:222 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NO:222 is fully modified with a PS backbone.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate. In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:222 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:222 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:222 is represented by SEQ ID NO:223: eTseGoeGseC*oeAsdAsdTsdUsdCsdTsdTsdTsdCsdTsdTseGoeAseAoeAseA (SEQ ID NO:223)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON92) (SEQ ID NO: 224)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:224. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:224 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:224 present in the gap of said oligonucleotide (C at position 7 and at position 1 1 of SEQ ID NO:224) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:224 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:224 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:224 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NO:224 is fully modified with a PS backbone.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate. In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:224 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:224 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:224 is represented by SEQ ID NO:225: eGseC*seAseAseTsdUsdC*sdTsdTsdTsdC*sdTsdTsdGsdAseAseAseAseTseG (SEQ ID NO:225)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON93) (SEQ ID NO: 226)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:226. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:226 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:226 present in the gap of said oligonucleotide (C at position 8 and at position 12 of SEQ ID NO:226) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:226 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:226 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:226 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NO:226 is fully modified with a PS backbone.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate. In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:226 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:226 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:226 is represented by SEQ ID NO:227: eGseGseC*seAseAsdTsdUsdC*sdTsdTsdTsdC*sdTsdTsdGseAseAseAseAseT (SEQ ID NO:227)
In an embodiment the antisense oligonucleotide comprises the following base sequence: TGGCAATUCTTTCTTGAAAA (AON94) (SEQ ID NO: 228)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:228. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:228 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:228 present in the gap of said oligonucleotide (C at position 9 and at position 13 of SEQ ID NO:228) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:228 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:228 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:228 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NO:228 is fully modified with a PS backbone.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate. In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:228 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:228 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:228 is represented by SEQ ID NO:229: eTseGseGseC*seAsdAsdTsdUsdC*sdTsdTsdTsdC*sdTsdTseGseAseAseAseA (SEQ ID NO:229) In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON95) (SEQ ID NO: 230)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:230. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NQ:230 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NQ:230 present in the gap of said oligonucleotide (C at position 7 and at position 1 1 of SEQ ID NQ:230) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NQ:230 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:230 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NQ:230 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NQ:230 is fully modified with a PS backbone.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate. In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:230 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:230 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NQ:230 is represented by SEQ ID NO:231 : eGseC*oeAoeAoeTsdUsdC*sdTsdTsdTsdC*sdTsdTsdGsdAseAoeAoeAoeTseG (SEQ ID NO:231)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON96) (SEQ ID NO: 232)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:232. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:232 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:232 present in the gap of said oligonucleotide (C at position 8 and at position 12 of SEQ ID NO:232) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:232 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:232 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:232 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NO:232 is fully modified with a PS backbone.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate. In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:232 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:232 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:232 is represented by SEQ ID NO:233: eGseGoeC*oeAoeAsdTsdUsdC*sdTsdTsdTsdC*sdTsdTsdGseAoeAoeAoeAseT (SEQ ID NO:233)
In an embodiment the antisense oligonucleotide comprises the following base sequence: TGGCAATUCTTTCTTGAAAA (AON97) (SEQ ID NO: 234)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:234. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:234 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:234 present in the gap of said oligonucleotide (C at position 9 and at position 13 of SEQ ID NO:234) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:234 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:234 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:234 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NO:234 is fully modified with a PS backbone.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate. In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:234 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:234 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:234 is represented by SEQ ID NO:235: eTseGoeGoeC*oeAsdAsdTsdUsdC*sdTsdTsdTsdC*sdTsdTseGoeAoeAoeAseA (SEQ ID NO:235) In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON98) (SEQ ID NO: 236)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:236. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:236 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:236 present in the gap of said oligonucleotide (C at position 7 and at position 1 1 of SEQ ID NO:236) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:236 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:236 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:236 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NO:236 is fully modified with a PS backbone. In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate. In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:236 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:236 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:236 is represented by SEQ ID NO:237: eGseC*oeAseAoeTsdUsdC*sdTsdTsdTsdC*sdTsdTsdGsdAseAoeAseAoeTseG (SEQ ID NO:237)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON99) (SEQ ID NO: 238)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:238. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:238 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:238 present in the gap of said oligonucleotide (C at position 8 and at position 12 of SEQ ID NO:238) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:238 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:238 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NO:238 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NO:238 is fully modified with a PS backbone.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate. In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:238 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:238 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:238 is represented by SEQ ID NO:239: eGseGoeC*seAoeAsdTsdUsdC*sdTsdTsdTsdC*sdTsdTsdGseAoeAseAoeAseT (SEQ ID NO:239)
In an embodiment the antisense oligonucleotide comprises the following base sequence: TGGCAATUCTTTCTTGAAAA (AON100) (SEQ ID NO: 240)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:240. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NQ:240 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NQ:240 present in the gap of said oligonucleotide (C at position 9 and at position 13 of SEQ ID NQ:240) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NQ:240 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:240 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). In an embodiment, the oligonucleotide comprising SEQ ID NQ:240 has 1 , 2 or 3 phosphosdiester linkages in each of its wings, preferably 3 phosphodiester linkages in each of its wings. In another embodiment, the oligonucleotide comprising SEQ ID NQ:240 is fully modified with a PS backbone.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate. In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:240 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:240 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NQ:240 is represented by SEQ ID NO:241 : eTseGoeGseC*oeAsdAsdTsdUsdC*sdTsdTsdTsdC*sdTsdTseGoeAseAoeAseA (SEQ ID NO:241)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON101) (SEQ ID NO: 242)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:242. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:242 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:242 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO:242) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:242 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:242 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:242 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:242 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:242 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:242 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 1 .
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:242 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi-Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:242 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:242 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE. A most preferred oligonucleotide comprising SEQ ID NO:242 is represented by SEQ ID NO:243: eGpNdmieC*oeAseAoeTsdUsdCsdTsdTsdTsdCsdTsdTsdGsdAseAoeAseAoeTpNdmieG (SEQ ID NO:243)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AON102) (SEQ ID NO: 244)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:244. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:244 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:244 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO:244) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:244 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:244 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:244 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:244 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:244 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:244 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:244 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi-Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:244 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:244 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:244 is represented by SEQ ID NO:245: eGpNdmieC*PNdmieApNdmieAoeT sdUsdCsdT sdT sdT sdCsdT sdT sdGsdAseAoeApNdmieApNdmieT PNdmieG (SEQ ID NO:245)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AQN103) (SEQ ID NO: 246)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:246. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:246 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:246 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO:246) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:246 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:246 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:246 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:246 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:246 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:246 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 2.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:246 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate. In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi-Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:246 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:246 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:246 is represented by SEQ ID NO:247: eGpNdmieC*oeApNdmieAoeT sdUsdCsdTsdT sdT sdCsdT sdT sdGsdAseAoeApNdmieAoeT PNdmieG (SEQ ID NO:247)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AQN104) (SEQ ID NO: 248)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:248. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:248 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:248 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO:248) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:248 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:248 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:248 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:248 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:248 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:248 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3. In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:248 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi-Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:248 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:248 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:248 is represented by SEQ ID NO:249: eGpNdmieC*oeApNdmieAoeT PNdmidUsdCsdTsdTsdTsdCsdTsdTsdGsdApNdmieAoeApNdmieAoeT PNdmieG (SEQ ID NO:249)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AQN105) (SEQ ID NO: 250)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:250. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NQ:250 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NQ:250 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NQ:250) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NQ:250 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:250 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NQ:250 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:250 is fully modified with a PS backbone. In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:250 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:250 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 2.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NQ:250 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi-Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:250 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:250 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NQ:250 is represented by SEQ ID NO:251 : eGpNdmieC*PNdmieAseAoeTsdUsdCsdTsdTsdTsdCsdTsdTsdGsdAseAoeAseApNdmieT PNdmieG (SEQ ID NO:251)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GCAATUCTTTCTTGAAAATG (AQN106) (SEQ ID NO: 252)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:252. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:252 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:252 present in the gap of said oligonucleotide (C at position 7 and position 11 of SEQ ID NO:252) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:252 are 5- methyl cytosines. In an embodiment, the oligonucleotide comprising SEQ ID NO:252 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:252 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:252 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:252 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:252 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:252 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has two distinct internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:252 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:252 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:252 is represented by SEQ ID NO:253: eGpNdmieC*PNdmieAseApNdmieTsdUsdCsdTsdTsdTsdCsdTsdTsdGsdAseApNdmieAseApNdmieT PNdmieG (SEQ ID NO:253)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AQN107) (SEQ ID NO: 254)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:254. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:254 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:254 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:254) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:254 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:254 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:254 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:254 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:254 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:254 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 1 .
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:254 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi-Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:254 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:254 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:254 is represented by SEQ ID NO:255: eGpNdmieGoeC*seAoeAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdGseAoeAseAoeApNdmieT (SEQ ID NO:255)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AQN108) (SEQ ID NO: 256)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:256. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:256 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:256 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:256) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:256 are 5-methylcytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:256 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:256 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:256 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:256 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:256 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:256 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi-Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:256 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:256 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:256 is represented by SEQ ID NO:257: eGpNdmieGpNdmieC*PNdmieAoeAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdGseAoeApNdmieApNdmieApNdmieT (SEQ ID NO:257)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON109) (SEQ ID NO: 258)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:258. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:258 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:258 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:258) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:258 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:258 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:258 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:258 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:258 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:258 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 2.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:258 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi-Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:258 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:258 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE. A most preferred oligonucleotide comprising SEQ ID NO:258 is represented by SEQ ID NO:259: eGpNdmieGoeC*PNdmieAoeAsdT sdUsdCsdT sdT sdT sdCsdT sdT sdGseAoeApNdmieAoeApNdmieT (SEQ ID NO:259)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON110) (SEQ ID NO: 260)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NQ:260. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NQ:260 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NQ:260 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NQ:260) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NQ:260 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NQ:260 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NQ:260 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:260 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NQ:260 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NQ:260 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NQ:260 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi-Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:260 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:260 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:260 is represented by SEQ ID NO:261 : eGpNdmieGoeC*PNdmieAoeApNdmidTsdUsdCsdTsdTsdTsdCsdTsdTsdGpNdmieAoeApNdmieAoeApNdmieT (SEQ ID NO:261)
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON111) (SEQ ID NO: 262)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:262. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:262 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:262 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:262) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:262 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:262 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:262 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:262 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:262 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:262 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 2.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:262 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi-Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:262 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:262 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:262 is represented by SEQ ID NO:263:
Figure imgf000133_0001
In an embodiment the antisense oligonucleotide comprises the following base sequence: GGCAATUCTTTCTTGAAAAT (AON112) (SEQ ID NO: 264)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:264. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:264 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:264 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NO:264) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:264 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:264 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:264 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:264 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:264 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:264 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:264 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has two distinct internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:264 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:264 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:264 is represented by SEQ ID NO:265: eGpNdmieGpNdmieC*seApNdmieAsdTsdUsdCsdTsdTsdTsdCsdTsdTsdGseApNdmieAseApNdmieApNdmieT (SEQ ID NO:265)
In an embodiment the antisense oligonucleotide comprises the following base sequence: TGGCAATUCTTTCTTGAAAA (AON113) (SEQ ID NO: 266)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:266. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:266 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:266 present in the gap of said oligonucleotide (C at position 9 and position 13 of SEQ ID NO:266) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:266 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:266 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:266 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:266 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:266 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:266 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 1 .
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:266 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi-Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:266 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:266 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:266 is represented by SEQ ID NO:267: eTpNdmieGoeGseC*oeAsdAsdTsdUsdCsdTsdTsdTsdCsdTsdTseGoeAseAoeApNdmieA (SEQ ID NO:267)
In an embodiment the antisense oligonucleotide comprises the following base sequence: TGGCAATUCTTTCTTGAAAA (AON114) (SEQ ID NO: 268)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:268. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:268 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:268 present in the gap of said oligonucleotide (C at position 9 and position 13 of SEQ ID NO:268) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:268 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:268 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:268 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:268 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:262 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:268 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:268 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi-Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:268 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:268 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:268 is represented by SEQ ID NO:269: eT PNdmieGpNdmieGpNdmieC*oeAsdAsdTsdUsdCsdTsdTsdTsdCsdTsdTseGoeApNdmieApNdmieApNdmieA (SEQ ID NO:269)
In an embodiment the antisense oligonucleotide comprises the following base sequence: TGGCAATUCTTTCTTGAAAA (AON115) (SEQ ID NO: 270)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:270. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NQ:270 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NQ:270 present in the gap of said oligonucleotide (C at position 8 and position 12 of SEQ ID NQ:270) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:270 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:270 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NQ:270 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NQ:270 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NQ:270 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NQ:270 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 2.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NQ:270 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi-Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NQ:270 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NQ:270 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NQ:270 is represented by SEQ ID NO:271 : eT PNdmieGoeGpNdmieC*oeAsdAsdT sdUsdCsdT sdT sdTsdCsdT sdTseGoeApNdmieAoeApNdmieA (SEQ ID NO:271)
In an embodiment the antisense oligonucleotide comprises the following base sequence: TGGCAATUCTTTCTTGAAAA (AON116) (SEQ ID NO: 272)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:272. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:272 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:272 present in the gap of said oligonucleotide (C at position 9 and position 13 of SEQ ID NO:272) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:272 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:272 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:272 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:272 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:272 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:272 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:272 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi-Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:272 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:272 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:272 is represented by SEQ ID NO:273: eT PNdmieGoeGpNdmieC*oeApNdmidAsdTsdUsdCsdTsdTsdTsdCsdTsdT PNdmieGoeApNdmieAoeApNdmieA (SEQ ID NO:273)
In an embodiment the antisense oligonucleotide comprises the following base sequence: TGGCAATUCTTTCTTGAAAA (AON117) (SEQ ID NO: 274)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:274. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:274 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:274 present in the gap of said oligonucleotide (C at position 9 and position 13 of SEQ ID NO:274) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:274 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:274 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:274 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:274 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:274 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:274 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 2.
In an embodiment, at least one internucleoside linkages in one wing (or in both wings) is not modified and is thus a phosphodiester internucleoside linkage. A preferred internucleoside linkage modification in one wing (or in both wings) is phosphorothioate
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:274 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has three distinct internucleoside linkages, preferably phosphodiester, dmi-Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:274 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:274 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE. A most preferred oligonucleotide comprising SEQ ID NO:274 is represented by SEQ ID NO:275: eT PNdmieGpNdmieGseC*oeAsdAsdTsdUsdCsdTsdTsdT sdCsdT sdTseGoeAseApNdmieApNdmieA (SEQ I D NO:275)
In an embodiment the antisense oligonucleotide comprises the following base sequence: TGGCAATUCTTTCTTGAAAA (AON118) (SEQ ID NO: 276)
In this embodiment, the antisense oligonucleotide has a length of 20 to 50 nucleotides and comprises the base sequence SEQ ID NO:276. The length may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
Each nucleotide may be a nucleotide analogue as identified earlier herein. If a base is modified or if a base analogue is being used, said modified base or base analogue should keep the same base pair specificity as the base it replaces.
In an embodiment, at least one C of the oligonucleotide comprising SEQ ID NO:276 is 5- methylcytosine. In another embodiment, the C of the oligonucleotide comprising SEQ ID NO:276 present in the gap of said oligonucleotide (C at position 9 and position 13 of SEQ ID NO:276) are not methylated. In another embodiment, all Cs of the oligonucleotide comprising SEQ ID NO:276 are 5- methyl cytosines.
In an embodiment, the oligonucleotide comprising SEQ ID NO:276 has at least one of its internucleoside linkages which is modified, preferably as a phosphorothioate or phosphoramidate (preferably mesyl-phosphoramidate). More preferably, the oligonucleotide comprising SEQ ID NO:276 has 1 , 2 or 3 phosphosdiesters linkage in each of its wings, preferably 3 phosphodiesters linkage in each of its wings. In an embodiment, the oligonucleotide comprising SEQ ID NO:276 is fully modified with a PS backbone.
In an embodiment, one internucleoside linkage modification of one wing or preferably of each of the wings of the oligonucleotide comprising SEQ ID NO:276 is a dmi-Phosphoramidate (PNdmi) linkage. Preferably, one wing or each of the wings of the oligonucleotide comprising SEQ ID NO:276 comprises 1 or 2 or 3 or 4 or 5 PNdmi linkages, more preferably 2 or 3 or 4, most preferably 3.
In an embodiment, one wing or preferably each wing of the oligonucleotide comprising SEQ ID NO:276 comprises two distinct modified internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment, the oligonucleotide has two distinct internucleoside linkages, preferably dmi- Phosphoramidate and phosphorothioate.
In an embodiment one wing or preferably each of the wings of the oligonucleotide comprising SEQ ID NO:276 has at least one internucleoside linkage modification and/or at least one sugar modification and/or at least one base modification compared to an RNA-based antisense oligonucleotide. In a preferred embodiment, one wing or more preferably both wings of the oligonucleotide comprising SEQ ID NO:276 comprises 1 , 2, 3, 4 or 5 nucleotides. In a more preferred embodiment, one wing or even more preferably both wings comprise 5 nucleotides. Most preferably both wings comprise 5 nucleotides and are MOE.
A most preferred oligonucleotide comprising SEQ ID NO:276 is represented by SEQ ID NO:277: eTpNdmieGpNdmieGseC*PNdmieAsdAsdTsdUsdCsdTsdTsdTsdCsdTsdTseGpNdmieAseApNdmieApNdmieA (SEQ ID NO:277)
Preferred oligonucleotides comprise SEQ ID NO: 2, 4, 6, 8, 7,8, 9, 10, 11 , 12, 14, 16, 18, 20, 22, 24, 25, 26, 27, 28, 29, 30, 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99, 101 , 103, 105, 107, 109, 111 , 113, 115, 117,122, 124, 126, 128,130,132,134,136,138,140,142,144, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 204, 217, 219, 221 , 223, 225, 227, 229, 231 , 233, 235, 237, 239, 241 , 243, 245, 247, 249, 251 , 253, 255, 257, 259, 261 , 263, 264, 265, 267, 269, 271 , 273, 275, 277 preferably SEQ ID NO: 7, 8, 9, 10, 11 , 12, 25, 26, 27, 28, 29, 30, 33, 34, 35, 36, 37, 38, 39, 47, 85, 97, 99, 115, 192, 194, 196, 198, 200, 221 , 223 more preferably SEQ ID NO: 8, 10, 12, 26, 28, 30, 35, 37, 39, 47, 85, 97, 99, 115, 221 , 223 and most preferably SEQ ID NO: 26, 28, 30, 35, 37, 39, 47, 85, 97, 99, 115, 221 , 223.
Composition
In a further aspect, there is provided a composition comprising the antisense oligonucleotide of the invention, wherein the composition comprises a pharmaceutically acceptable excipient. This composition preferably comprises or consists of or essentially consists of an antisense oligonucleotide as described above.
All preferred features relating to each of these antisense oligonucleotides have been disclosed in the section entitled “antisense oligonucleotide”.
In a preferred embodiment, said composition is for use as a medicament. Said composition is therefore a pharmaceutical composition. A pharmaceutical composition usually comprises a pharmaceutically accepted carrier, diluent and/or excipient. In a preferred embodiment, a composition of the current invention comprises an antisense oligonucleotide as defined herein and optionally further comprises a pharmaceutically acceptable formulation, filler, preservative, solubilizer, carrier, diluent, excipient, salt, adjuvant and/or solvent. Such pharmaceutically acceptable carrier, filler, preservative, solubilizer, diluent, salt, adjuvant, solvent and/or excipient may for instance be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000.
A pharmaceutical composition may be further formulated to further aid in enhancing the stability, solubility, absorption, bioavailability, pharmacokinetics and cellular uptake of said antisense oligonucleotide, in particular formulations comprising excipients capable of forming complexes, nanoparticles, microparticles, nanotubes, nanogels, hydrogels, poloxamers or pluronics, polymersomes, colloids, microbubbles, vesicles, micelles, lipoplexes, and/or liposomes. Examples of nanoparticles include polymeric nanoparticles, gold nanoparticles, magnetic nanoparticles, silica nanoparticles, lipid nanoparticles, sugar particles, protein nanoparticles and peptide nanoparticles.
A preferred composition comprises at least one excipient that may further aid in enhancing the targeting and/or delivery of said antisense oligonucleotide to the central nerve system, and/or the spinal cord, and/or into brain tissue and/or to a neuronal tissue and/or a cell. A cell may be a neuronal cell.
Many of these excipients are known in the art (e.g. see Bruno, 2011) and may be categorized as a first type of excipient. Examples of first type of excipients include polymers (e.g. polyethyleneimine (PEI), polypropyleneimine (PPI), dextran derivatives, butylcyanoacrylate (PBCA), hexylcyanoacrylate (PHCA), poly(lactic-co-glycolic acid) (PLGA), polyamines (e.g. spermine, spermidine, putrescine, cadaverine), chitosan, poly(amido amines) (PAMAM), poly(ester amine), polyvinyl ether, polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG) cyclodextrins, hyaluronic acid, colominic acid, and derivatives thereof), dendrimers (e.g. poly(amidoamine)), lipids {e.g. 1 ,2-dioleoyl-3-dimethylammonium propane (DODAP), dioleoyldimethylammonium chloride (DODAC), phosphatidylcholine derivatives [e.g 1 ,2- distearoyl-sn-glycero-3-phosphocholine (DSPC)], lyso-phosphatidylcholine derivaties [e.g. 1- stearoyl-2-lyso-sn-glycero-3-phosphocholine (S-LysoPC)], sphingomyeline, 2-{3-[bis-(3-amino- propyl)-amino]-propylamino}-N-ditetracedyl carbamoyl methylacetamide (RPR209120), phosphoglycerol derivatives [e.g. 1 ,2-dipalmitoyl-sn-glycero-3-phosphoglycerol sodium salt (DPPG-Na), phosphaticid acid derivatives [1 ,2-distearoyl-sn-glycero-3-phosphaticid acid, sodium salt (DSPA), phosphatidylethanolamine derivatives [e.g. dioleoyl-phosphatidylethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),2-diphytanoyl-sn-glycero-3- phosphoethanolamine (DPhyPE),], N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA), 1 ,3-di-oleoyloxy-2- (6-carboxy-spermyl)-propylamid (DOSPER), (1 ,2-dimyristyolxypropyl-3-dimethylhydroxy ethyl ammonium (DMRIE), (N1 -cholesteryloxycarbonyl-3, 7-diazanonane-1 ,9-diamine (CDAN), dimethyldioctadecylammonium bromide (DDAB), 1-palmitoyl-2-oleoyl-sn-glycerol-3- phosphocholine (POPC), (b-L-arginyl-2,3-L-diaminopropionic acid-N-palmityl-N-olelyl-amide trihydrochloride (AtuFECTOI), N,N-dimethyl-3-aminopropane derivatives [e.g. 1 ,2-distearoyloxy- N,N-dimethyl-3-aminopropane (DSDMA), 1 ,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DoDMA), 1 ,2-dilinoleyloxy-N,N-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4- dimethylaminomethyl [1 ,3]-dioxolane (DLin-K-DMA), phosphatidylserine derivatives [1 ,2-dioleyl- sn-glycero-3-phospho-L-serine, sodium salt (DOPS)], cholesteroljproteins (e.g. albumin, gelatins, atellocollagen), and peptides (e.g. protamine, PepFects, NickFects, polyarginine, polylysine, CADY, MPG).
Another preferred composition may comprise at least one excipient categorized as a second type of excipient. A second type of excipient may comprise or contain a conjugate group as described herein to enhance targeting and/or delivery of the antisense oligonucleotide of the invention to a tissue and/or cell and/or into a tissue and/or cell, as for example neuronal tissue or cell. Neuronal tissues or cells may be all the neuronal tissues or cells known to be affected in FAD. Such neuronal tissues or cells may be derived from or may be from the hippocampus, amygdala, cerebellar cortex (frontal lobe, parietal lobe, temporal lobe, occipital lobe), cerebellum, corpus callosum or the thalamus, beyond others. Both types of excipients may be combined together into one single composition as identified herein. A conjugate group may be selected from a ligand, including transferrin-receptor targeting moieties like antibody fragments, aptamers, peptides, sugars or lipids, ethylene glycol moieties.
The skilled person may select, combine and/or adapt one or more of the above or other alternative excipients and delivery systems to formulate and deliver an antisense oligonucleotide for use in the present invention.
Such a pharmaceutical composition of the invention may be administered in an effective concentration at set times to an animal, preferably a mammal. More preferred mammal is a human being. An antisense oligonucleotide or a composition as defined herein for use according to the invention may be suitable for direct administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing a disease or condition as identified herein, and may be administered directly in vivo, ex vivo or in vitro. Administration may be via systemic and/or parenteral routes, for example intravenous, subcutaneous, intraventricular, intrathecal, intra- cerebrospinal, intramuscular, intranasal, enteral, intravitreal, intracerebral, epidural or oral route.
Preferably, such a pharmaceutical composition of the invention may be encapsulated in the form of an emulsion, suspension, pill, tablet, capsule or soft-gel for oral delivery, or in the form of aerosol or dry powder or device for delivery to the cerebrospinal fluid.
In an embodiment, the pharmaceutical composition is a cerebrospinal composition or an artificial cerebrospinal composition also called an artificial cerebrospinal fluid (aCSF). Such composition comprises a buffer solution representative of the cerebrospinal fluid. Preferably such composition comprises the following ion concentrations: Na+ 150 mM, K+ 3.0 mM, Ca2+ 1.4 mM, Mg2+ 0.8 mM, P 1.0 mM and Cl- 155 mM. It is commonly used as a vehicle solution for administration of therapeutic agents to the central nervous system (CNS). The exact composition may slightly differ between suppliers. The oligonucleotide may be formulated in such composition and may be delivered using a device.
In an embodiment an antisense oligonucleotide of the invention may be used together with another compound already known to be used for the treatment of said disease. Such combined use may be a sequential use: each component is administered in a distinct composition. Alternatively, each compound may be used together in a single composition.
In a further aspect, there is therefore provided an antisense oligonucleotide or a composition for use as a medicament. In an embodiment, an antisense oligonucleotide or a composition for use as a medicament is preferably for treating and/or delaying and/or curing and/or preventing and/or ameliorating a disease or condition associated with an abnormal processing (or altered processing) of the Amyloid Precursor Protein (APP), preferably familiar Alzheimer disease (FAD). In such disease or condition with an abnormal processing (or altered processing) of the APP, one allele of a protein of the y-secretase complex is mutated compared to the wild-type protein (human PSEN1 protein is represented by SEQ ID NO:153 or 159 and is encoded by SEQ ID NO:152 or 169 and genomic DNA is represented by SEQ ID NO:151). In an embodiment, an allele of PSEN1 , preferably an allele of human PSEN1 is mutated. Known mutations have been listed earlier herein.
A subject or a patient having a disease or condition associated with an abnormal processing of the APP may be identified as such (may be diagnosed) by the presence of at least one of the following symptoms and/or parameters:
• increased A042/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably increased Ap 42/40 and/or Ap 42/38 ratios, more preferably an increased Ap42/38 ratio) in the plasma of symptomatic FAD patients,
• increased Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably increased Ap 42/40 and/or Ap 42/38 ratios, more preferably an increased Ap42/38 ratio) in the cerebrospinal fluid or plasma of FAD patients with no detectable Ap plaques in the brain (i.e. so called asymptomatic patients)
• decreased Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably decreased Ap 42/40 and/or Ap 42/38 ratios, more preferably a decreased Ap42/38 ratio) in the cerebrospinal fluid of patients with Ap plaques in the brain
• presence of Ap plaques in the brain (measured with positron emission tomography (PET) using ligands specific for fibrillar Ap)
• an increased level of Tau and/or phosphorylated Tau 181 (p-Tau181) in the cerebrospinal fluid and the plasma
• presence of neurofibrillary tangles in the brain (measured with positron emission tomography (PET) using ligands specific for Tau)
• reduce of cerebral metabolic rate for glucose (assessed with in vivo imaging using 18[F]fluoro-2-deoxy-D-glucose PET (FDG-PET)
• neuronal degeneration and especially an atrophy or degeneration of at least one of the following brain regions: hippocampus, amygdala, cortex, cerebellum, frontal lobe, parietal lobe, temporal lobe, occipital lobe, frontal lobe, corpus callosum and the thalamus assessed with neuroimaging techniques such as MRI
• an increased SNAP25 level in cerebrospinal fluid • an increased level of neurofilament light (NFL) in cerebrospinal fluid and the plasma
• cognitive impairments (assessed by MMSE and Alzheimer's Disease Assessment Scale-Cognitive subscale)
In this context, increased Ap 42/40 and/or Ap 42/38 ratios has been already defined earlier herein.
In this context, an increased level of Tau and/or phosphorylated Tau 181 (p-Tau181) in the cerebrospinal fluid and/or the plasma may mean an increased level of Tau and/or phosphorylated Tau 181 (p-Tau181) which is increased of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to its level in a healthy subject. The level of Tau may be assessed by techniques known to the skilled person. In an embodiment it is assessed using ELISA, preferably as carried out in the experimental part. Level of Tau and neurofibrillary tangles formed by Tau aggregations may also be assessed using PET imaging.
An atrophy or degeneration of a brain region and especially of at least one of the following brain regions listed above may be assessed by a physician using common known techniques such as MRI imaging.
In this context, a decreased metabolic rate for glucose in the brain may mean a decreased level of metabolic rate which is decreased of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to its level in a healthy subject. The metabolic rate for glucose in the brain may be assessed by techniques known to the skilled person.
In this context, neuronal degeneration and especially an atrophy or degeneration of at least one of the following brain regions: hippocampus, amygdala, cortex, cerebellum, frontal lobe, parietal lobe, temporal lobe, occipital lobe, frontal lobe, corpus callosum and the thalamus may be assessed with neuroimaging techniques such as MRI. For example to investigate neurodegeneration we may use the hippocampal and ventricular volumes as a parameter. The white matter hyperintensity (WMH) volume, a cerebrovascular disease marker may also be used.
In this context, an increased level of NFL in the cerebrospinal fluid and the plasma is indicative of a disease or condition associated with neurodegeneration. Increased NFL levels may mean an increased level of NFL which is increased of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to its level in a healthy subject. The level of NFL may be assessed by techniques known to the skilled person. In an embodiment it is assessed using ELISA. In this context, an increased level of SNAP25 in the cerebrospinal fluid is indicative of a disease or condition associated with synaptic damage and neurodegeneration. Increased SNAP25 levels may mean an increased level of SNAP25 which is increased of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to its level in a healthy subject. The level of SNAP25 may be assessed by techniques known to the skilled person. In an embodiment it is assessed using ELISA.
In an embodiment, an antisense oligonucleotide or a composition for use according to the invention is able to induce a therapeutic effect. A therapeutic effect may mean that the antisense oligonucleotide elicits at least one of the following:
• the oligonucleotide is able to preferentially silence, inactivate, break down, knock down, reduce and/or decrease (the levels of) mutated PSEN1 or PSEN2 transcripts (preferably PSEN1 transcripts) and preferably the oligonucleotide is not (or minimally) able to silence, inactivate, break down, knock down, reduce and/or decrease (the levels of) wild-type PSEN1 or PSEN2 transcripts (preferably PSEN1 transcripts),
• the oligonucleotide is able to normalize, correct, or reverse abnormal A042/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably Ap42/38 and/or AB43/40 ratios, more preferably the Ap42/38 ratio), and
• the oligonucleotide is able to alleviate one or more symptom(s) and/or characteristic(s) and/or to improve a parameter linked with or associated with a disease or condition associated with an abnormal processing of the Amyloid Precursor Protein (APP) preferably familiar Alzheimer disease (FAD).
The silencing, inactivation, knocking down, breaking down, decrease or the reduction of the (levels of) the mutated allele (transcript and protein) has already been defined earlier herein. It corresponds to the first way of assessing an activity of the antisense oligonucleotide of the invention.
The normalization, correction, or reversion of abnormal Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably Ap42/38 and/or Ap43/40 ratios, more preferably the Ap42/38 ratio) may mean a decrease of the Ap42/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios (preferably Ap42/38 and/or Ap43/40 ratios, more preferably the Ap42/38 ratio) as earlier defined herein. It corresponds to the second way of assessing an activity of the oligonucleotide of the invention. The alleviation of one or more symptom(s) and/or characteristic(s) and/or the improvement of the value of a parameter linked with or associated with a disease or condition associated with an abnormal processing of the Amyloid Precursor Protein (APP) (preferably familiar Alzheimer disease (FAD)) may refer to at least one of the following: a decreased level of Tau and/or phosphorylated Tau 181 in the cerebrospinal fluid and plasma, a metabolism of glucose in the cortex which is increased, a neuronal degeneration which is delayed or reversed and especially a neuronal degeneration of at least one of the following brain regions is delayed or reversed: hippocampus, amygdala, cortex, cerebellum, frontal lobe, parietal lobe, temporal lobe, occipital lobe, frontal lobe, corpus callosum and the thalamus, an decreased SNAP25 level in cerebrospinal fluid, a decreased level of NFL in cerebrospinal fluid and/or plasma, cognitive impairments that are reversed that are improved. a decrease I slowdown the formation of Ap plaques formation in the brain.
In this context, a decreased level of Tau and/or phosphorylated Tau 181 in the cerebrospinal fluid and plasma may mean a decreased level of Tau of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to its level at the onset of the treatment. The level of Tau and phosphorylated Tau 181 may be assessed by techniques known to the skilled person as earlier defined herein.
An atrophy or degeneration of a brain region and especially of at least one of the brain regions listed above may be assessed as earlier defined herein.
In this context, a decreased level of NFL may mean a decrease of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to its level at the onset of the treatment. The level of NFL may be assessed by techniques known to the skilled person as earlier defined herein.
In this context, a decreased level of SNAP25 may be of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to its level at the onset of the treatment. The level of SNAP25 may be assessed by techniques known to the skilled person as earlier defined herein.
In this context, a decrease / slowdown of the formation of A plaques formation in the brain may mean a decrease of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to its level at the onset of the treatment. Ap plaques formation in the brain may be monitored using techniques known to the skilled person. Preferred techniques have been earlier defined herein.
An antisense oligonucleotide as defined herein is able to improve one parameter or reduce a symptom or characteristic if after at least one week, one month, six month, one year or more of treatment using a dose of said antisense oligonucleotide of the invention as identified herein said parameter is said to have been improved or said symptom or characteristic is said to have been reduced.
Improvement in this context may mean that said parameter had been significantly changed towards a value of said parameter for a healthy person and/or towards a value of said parameter that corresponds to the value of said parameter in the same individual at the onset of the treatment.
Reduction or alleviation in this context may mean that said symptom or characteristic had been significantly changed towards the absence of said symptom or characteristic which is characteristic for a healthy person and/or towards a change of said symptom or characteristic that corresponds to the state of the same individual at the onset of the treatment.
In a further aspect, there is provided the use of a composition or an antisense oligonucleotide as described in the previous sections for use as a medicament or part of therapy, or applications in which said oligonucleotide exerts its activity intracellularly.
Preferably, an oligonucleotide or composition of the invention is for use as a medicament or part of a therapy for preventing, delaying, curing, ameliorating and/or treating a disease or condition associated with an abnormal processing of the Amyloid Precursor Protein (APP), preferably familiar Alzheimer disease (FAD).
In a further aspect there is provided a use of a composition or an antisense oligonucleotide as described in the previous sections, for the manufacture of a medicament for preventing, delaying, curing, ameliorating and/or treating a disease or condition associated with an abnormal processing of the Amyloid Precursor Protein (APP), preferably familiar Alzheimer disease (FAD). In a further aspect, there is provided a method for preventing, treating, curing, ameliorating and/or delaying a condition or disease as defined in the previous section in an individual, in a cell, tissue or organ of said individual. The method comprises administering an antisense oligonucleotide or a composition of the invention to said individual or a subject in the need thereof. In some embodiments, administering an antisense oligonucleotide or a composition means administering to a subject such as a subject in need thereof. In a preferred embodiment, a therapeutically effective amount of an antisense oligonucleotide or a composition is administered.
The method according to the invention wherein an antisense oligonucleotide or a composition as defined herein may be suitable for administration to a cell, tissue and/or an organ in vivo of individuals affected by any of the herein defined diseases or at risk of developing said disease, and may be administered in vivo or in vitro. An individual or a subject in need is preferably a mammal, more preferably a human being.
In an embodiment, in an in vitro method of the invention (such as in gymnotic uptake experiment), a concentration of an oligonucleotide or composition is ranged from 0.1 nM to 5 pM or from 1 nM to 25 pM, or from 2 nM to 10 pM.
In an embodiment, in an in vivo method of the invention , a concentration of an oligonucleotide or composition is ranged from 0.1 pM to 10 mM or from 0.3 pM to 1 mM, or from 0.5 pM to 50 mM or from 1 pM to 10 mM.
Dose ranges of an antisense oligonucleotide or composition according to the invention are preferably designed on the basis of ascending dose studies in clinical trials (/n vivo use) for which rigorous protocol requirements exist.
In an intrathecal (IT) or intracerebroventricular (ICV) (i.e. administration into the central nervous system) delivery, an antisense oligonucleotide as defined herein may be used at a dose which is ranged from 10 to 300 pg/dose in mice or 1-60 mg in Non-Human Primates (NHPs), 1-200 or 2- 150 pr 5-120 mg in humans. The frequency of the IT or ICV dose is once weekly, biweekly, or once per 4, 8. 12, 16, 20 or 24 weeks.
In an SC (subcutaneous) or IV (intravenous) delivery, an antisense oligonucleotide as defined herein may be used at a dose which is ranged from 1 to 500 or 3-300 mg/kg in mice, 0.1 to 100 or 0.5 to 75 or 1-50 mg/kg in NHPs, 20-500 or 30-450 or 50-400 mg in humans. The frequency of the SC or IV treatment is daily, every 2 days, every 4 days, twice weekly, weekly, biweekly, or once per month.
The ranges of concentration or dose of antisense oligonucleotide or composition as given above are preferred concentrations or doses for in vitro or in vivo uses. The skilled person will understand that depending on the identity of the antisense oligonucleotide used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration or dose of antisense oligonucleotide used may further vary and may need to be optimised any further.
In this document and in its claims, the verb "to comprise" and its conjugations is used in its nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. The verb “to comprise” is synonymous with the verb “to have” unless otherwise indicated. In addition the verb “to consist” may be replaced by “to consist essentially of’ meaning that an antisense oligonucleotide or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional components) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".
Each embodiment as identified herein may be combined together unless otherwise indicated. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
Table 1a: summary of the sequences identified in the application (part 1)
Figure imgf000151_0001
Table 1 b: summary of the sequences identified in the application (part 2)
Figure imgf000151_0002
Example 1
Introduction
Mutations in Psenl , like A431 E, affect the activity of y-secretase. This is a heterotetrameric complex in which presenilins act as the catalytic subunit in the cleavage of the APP C-terminal fragment (CTF). Impaired Psenl , leading to a reduced processivity of y-secretase, results in increase generation of long >Ap42 species at expenses of short <Ap38 species. This leads to an abnormal Ap profile, followed by amyloid deposition that causes synaptic dysfunction and neurodegeneration (Petit D et al., Mol Psychiatry. 2022 Apr 1). The therapeutic strategy of this invention aims at the specific reduction of mutant Psenl and normalization of Ap peptide profiles. To demonstrate the effectivity of a mouse sequence-specific AON of the invention, neuronal cell cultures derived from the Psenl A431 E;APPKi knock-in mouse model (Tg/Tg) were treated with AON20 (SEQ ID NO:118) for eight days. The resulting PSEN1 protein levels were assessed, as well as the y -secretase processivity and soluble Ap peptides profiles.
Material and Methods
AO Ns All antisense oligonucleotides (AONs) have a phosphorothioate (PS) backbone with a DNA gap flanked by 2’-MOE wings at 5’ and 3’ end (Table 1). The AONs were synthesized in 5 pmol scale using a MerMade 12 synthesizer (Bioautomation) and standard phosphoramidite protocols. The AONs were cleaved and deprotected in a two-step sequence (DEA followed by cone. NH4OH treatment), purified by anion-exchange chromatography, desalted by size exclusion chromatography and lyophilized. Mass spectrometry confirmed the identity of all AONs, and purity (determined by UPLC) was found acceptable for all AONs (>80%).
Table 2: Mouse AON sequences
Figure imgf000152_0001
A=adenosine; G=guanine; T=thymine; C=cytosine; C*=5-methylcytosine. d=DNA, e=2’-MOE, s=PS linkage.
Corresponding non modified sequence of the oligonucleotide represented by SEQ ID NO: 118 is SEQ ID NO:119 (which is AGCGCCGGCAATTCTTTCTTG)
Corresponding non modified sequence of the oligonucleotide represented by SEQ ID NO: 120 is SEQ ID NO:157 (which is AGCGCCGGCAATTCTTTCTTG)
Primary mouse neuronal cultures (NCs)
The Psenl A431 E (GCG>GAA) knock-in mouse model was generated using CrisprCas9 technology. Two additional silent mutations were introduced in the target region, resulting in four nucleotide differences compared to the wild-type allele sequence. Psenl A431 E mice were subsequently crossed with mice carrying two copies of humanized amyloid-beta precursor protein APPKi mouse (Serneels et al., 2020). To obtain primary neuronal cell cultures, cerebral cortices from Psenl A431 E;APPKi E14 embryos were dissected. Tissue was dissociated by 15 min incubation with 0.25% trypsin in HBSS followed by mechanical dissociation using a fire polish Pasteur pipette. Cells were then seeded in poly-L-lysine-coated 96 well plates in MEM medium with 10% Horse serum, 0.6% D-Glucose and 1 % Pen-Strep. After 4h, medium is changed to Neurobasal medium supplemented with B27, 0.5 nM Glutamine and 1 % Pen-Strep. Half-medium was refreshed daily.
Gymnotic uptake
Primary neuronal cultures derived from PSEN1 A431 E homozygous embryos (psen1A431E/ A431E; APPKi embryos) were treated daily by addition of 0-1-10 |j.M AON (Table 2) to the medium, starting at day one after culture initiation. Cells were harvested after 8 days of treatment.
Protein extraction and Western blot Proteins were extracted in lysis buffer containing STE (NaCI/Tris-HCl/EDTA), 1 % Triton 100 and a cocktail of Proteases inhibitors. Protein concentrations were determined by the Bio-Rad DC protein assay (Bio-Rad). Samples were separated by SDSPAGE (4-12% Bis-Tris NuPAGE gels in MES running buffer, Life Technologies) and transferred onto nitrocellulose membranes (Life Technologies). After blocking in 4% non-fat milk, membranes were incubated overnight at 4°C with primary antibodies (Table 3) followed by washing and incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1 h, room temperature). After final washing, immunodetection was done using enhanced chemiluminescence (Western Lightning-Plus ECL, PerkinElmer), and immunoreactive protein bands were digitally captured and quantified on a Fuji MiniLAS 3000 imager (Fuji, Dusseldorf, Germany) using Image Studio Light software version 5.2 (LI-COR Biosciences, Cambridge, UK).
Table 3: Primary antibodies.
Figure imgf000153_0001
Quantification of soluble AB38, AB40 and AB42:
Ap38, Ap40 and Ap42 levels were measured in conditioned medium with either single-spot or multi spot 96 well plates pre-coated with neo epitope anti-Ap38, anti-Ap40, or -Ap42 antibodies using MSD technology. MSD antibody coated plates were blocked with 150 pl/well PBS buffer supplemented with 0.1 % casein for 1.5 hr at room temperature (600 rpm) and rinsed 5 x with 200 pl/well washing buffer (PBS + 0.05% Tween-20). 25 pL of SULFO-TAG labeled detection antibody against the N-terminus of human Abeta diluted in blocking buffer was mixed with 25 pL of standards (synthetic human Ap38, Ap40, Ap42 peptides) or samples diluted in blocking buffer and loaded 50 pL per well. After overnight incubation at 4°C plates were rinsed with washing buffer and 150 pl/well of the 2x MSD Read Buffer T was added. Plates were read immediately on MSD Sector Imager 6000.
Results
Treatment with AON 20 (SEQ OD NO: 118, Table 2) resulted in a dose-dependent reduction of full-length mutant PSEN1 , which reached 56% reduction at the highest 10 .M AON concentration (Figure 1A). To investigate whether the observed reduction in PSEN1 protein levels leads to a reduction in the y-secretase activity, the proteolytic processing of APP was assessed by quantifying the levels of APP C-terminal fragments (CTF), which is a direct substrate of the y- secretase complex. Western blot analysis revealed an accumulation of APP CTFs in neurons treated with AON 20 (Figure 1 B). Consequently, the levels of soluble Ap peptides were reduced, the Ap38 levels even down to 50% (Figure 1 C). Noteworthy, the Ap42/Ap38 ratio was not modified by the AON treatment (Figure 1 D).
Conclusions
AON 20, an AON of the invention, is effective in reducing mouse A431 E mutant Psenl protein levels. The downstream effect is shown by an increase in APP CTF and a decrease in Ap peptide levels.
Example 2
Introduction
Mutations in Psenl , like A431 E, reduce the functionality of y-secretase and increase the production of long Ap species (>Ap42) which leads to an abnormal Ap profile with increased Ap42/40 and Ap42/38 ratios (Szaruga M et al., Cell. 2017 Jul 27;170(3):443-456.e14, Petit D et al., Mol Psychiatry. 2022 Apr 1). The majority of FAD patients carry one Psenl mutant allele and one Wt allele. The therapeutic strategy of this invention aims at the specific reduction of mutant Psenl to allow replacement by Wt Psenl in the y-secretase complex, and to restore normal Ap peptides profiles. To investigate if the presence of the Wt Psenl protein can indeed normalize the Ap peptide profile and reduce the Ap42/Ap38 ratio, the Example 1 experiment was repeated, but with a different AON of the invention (AON 21 , SEQ ID NO:120 of Table 2) and using primary neurons derived from Psenl A431 E heterozygous mouse embryos (Psen1wt/ A431E; APPKi). Neurons were treated with AON 21 for 7 consecutive days and the levels of soluble Ap38, Ap40, and Ap42 peptides in the medium were measured as a read-out of y-secretase processivity.
Material and Methods
See Example 1 .
Results
Treatment with AON 21 reduced the levels of the three Ap peptides analysed (27% reduction of Ap38, 50% of Ap40, and 60% of Ap42 compared to the untreated condition (Figure 2A). Importantly, these changes in Ap levels also translated into a 45% reduction of the Ap42/Ap38 ratio in treated heterozygous neurons (Figure 2B), which suggests a shift in the production of Ap species towards the generation of shorter Ap38 peptides instead of Ap42 peptides. This was not observed in the psen1A431/A431E; APPKi cells (Example 1), which is hypothesized to relate to the presence of Wt Psenl protein, replacing the mutant Psenl in the y-secretase complex. Since Ap plaques are mainly formed by Ap42 peptide accumulation (Gravina et al., JBC. 1995: 270(13):7013-7016), such corrective effect is anticipated to slow down the progression of the disease.
Conclusions
AON21 , an AON of the invention, is effective in reducing long Ap peptide levels and improving the Ap42/Ap38 ratio. Example 3
Introduction
The majority of FAD patients carry one PSEN1 mutant allele and one Wt allele. The therapeutic strategy of this invention aims at the specific reduction of mutant PSEN1 , with limited impact on expression of the Wt protein. The human PSEN1 A431 E mutation is a C>A single nucleotide substitution. Compared to the A431 E mouse target sequence of Examples 1 and 2, containing four base substitutions compared to the Wt sequence, the design of human A431 E-specific AONs thus requires a higher level of fine-tuning chemical modifications and/or the position of the nucleotide opposite the mutation, to ensure maximum specificity for the mutated allele. A series of AONs was designed and initially screened for their activity versus specificity in fibroblasts transfected with plasmids containing either the Wt or A431 E mutant PSEN1 cDNA construct. The goal of this experiment was to pre-select AONs with most favourable activity/specificity profiles to be further screened in iPSC derived neurons from FAD patients with the A431 E mutation (see Example 5).
Material and Methods:
AONs
AONs specifically targeting the human A431 E PSEN1 mRNA were designed (see Table 4). AONs had a backbone containing phosphorothioate (PS), a phospodiester (PO), dmi-Phosphoramidate (PNdmi) and/or a mesyl-phosphoramidate (PNms) with a DNA gap flanked by 2’-MOE wings at 5’ and 3’ end. The AONs were synthesized in 5 pmol scale by a MerMade 12 synthesizer (Bioautomation) using standard phosphoramidite protocols. The AONs were cleaved and deprotected in a two-step sequence (DEA followed by cone. NH4OH treatment), purified by anion- exchange chromatography, desalted by size exclusion chromatography and lyophilized. Mass spectrometry confirmed the identity of all AONs, and purity (determined by UPLC) was found acceptable for all AONs (>80%).
Table 4: Human AON sequences
Figure imgf000156_0001
Figure imgf000157_0001
Figure imgf000158_0001
Figure imgf000159_0001
Figure imgf000160_0001
A=adenosine; G=guanine; T=thymine; C=cytosine; U=Uracil; C*=5-methylcytosine. D=DNA, e=2’-MOE, m= 2’-OMe, s=PS linkage, o = PO linkage, PNms = PNms linkage, PNdmi = PNdmi linkage. The corresponding sequences of the non-modified oligonucleotides are represented by SEQ ID NO:1 , 3, 5, 7, 9, 11 , 13,15, 17, 19, 21 , 23, 25, 27, 29, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 1 10, 1 12, 1 14, 116, 121 , 123, 125, 127, 129, 131 , 133, 135, 137, 139, 141 , 143, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260,262, 264, 266, 268, 270, 272, 274, 276.
Transfection of human PSEN1 expression vectors and AONs
NIH3T3 cells were cultured to 80% confluency in 6-wells plate and transfected with 2 pg of plasmid encoding the human wild-type (Wt) PSEN1 variant 1 (NM_000021 .4) or mutant A431 E PSEN1 transcripts with Flag-tags at the N-terminus. These plasmids also express Neomycin Phosphotransferase II (NPT-II). Three hours after plasmid transfection, cells were transfected with 10nM AON. Transfections were done using Lipofectamine 2000 and following manufacturer’s instructions. Cells were harvested 24 hours post AON transfection.
Protein extraction and WES:
Proteins were extracted from cell pellets using PLB-G Lysis buffer (16% SDS, 0.07 M Tris-HCI pH 6.8, 21 % Glycerol, 50mM DTT, and 1X proteases Inhibitor Cocktail) and mechanical dissociation with a syringe. Protein concentration was measured with Pierce 660nm Protein Assay Reagent (Thermo) supplemented with Ionic Detergent Compatibility Reagent (Thermo). Protein levels of Flag-PSEN1 and NPT-II were quantified using WES technology (Protein simple) according to the manufacturer’s instructions. The Jess/Wes Separation 12-230 kDa kit (Protein simple) was used. The following primary antibodies and concentrations were used: anti Flag-tag antibody (MAB8529, R&D Systems, 1/1000) and anti-NPT-ll antibody (NB110-60487, Bio- Techne, 1/2500). For correct NPT-II detection, anti-Mouse IgG H&L (ab6708, Abeam) was added to the anti-mouse Secondary antibody at 1/100 dilution. For each sample, the area of the Flag peak was divided by the area of the NPT-II peak to normalise each sample for plasmid transfection efficacy. Subsequently, the Flag/NPT-ll ratio of each treated sample was normalised to the ratio of the control untreated sample, which was transfected with the plasmid but not with the AON.
Results
A first series of AONs was tested wherein sequence and the position of the nucleotide opposite the mutation shifted from the 5’ end to the 3’ end (Table 4, Figure 3A). Results indicated that the best efficacy and selectivity towards the mutant A431 E PSEN1 was obtained with AONs wherein the mutational position is within the 3 first positions of the central part of the antisense oligonucleotide starting from 5’ to 3’ (i.e., in position 6, 7 or 8 of the oligonucleotide sequence starting from 5’ to 3’)(AON4, AON5, AON6, SEQ ID NO:8, 10, 12). AON4 and AON5 resulted in a 70% reduction of the mutant A431 E PSEN1 ratio, while this ratio is reduced up to 80% with AON6. In comparison, the reduction of the WT PSEN1 ratio reached 11 % with AON4, 30% with AON5 and 26% with AON6. No significant reduction was observed in cells treated with a scrambled AON compared to the AON untreated sample (Figure 3A).
In a second series, AON variants of AON4, AON5 and AON6 were designed containing a T (thymine) to U (uracil) substitution at the position opposite the mutation (Table 4, Figure 3B). This resulted in a higher specificity for the mutant allele: AON13, AON14 and AON15 (SEQ ID NO:26, 28, 30) reduced PSEN1 A431 E protein levels to similar levels as AON4, AON5 and AON6 (70%, 76% or 81 %, respectively), but the reduction of the WT PSEN1 protein levels was less: 5% for AON13 and AON15, and 20% for AON14 (Figure 3B).
In a third series, AON variants of AON14 (SEQ ID NO:28) were designed in which some PS linkages were substituted for PO, PNdmi, and/or PNms linkages (Figure 3C). We hypothesized that the reduction of PS content might reduce AON in-vivo toxicity (Moazami, et al., BioRxiv2021). The addition of PNdmi or PNms might increase AON potency and stability (Anderson BA et al., Nucleic Acids Res. 2021 Sep 20;49(16):9026-9041 . Kandasamy P et al., Nucleic Acids Res. 2022 Feb 2), while the incorporation of PO is predicted to increase the Tm which might result in a partial loss of AON allele specificity due to stronger binding to WT PSEN1 mRNA. Surprisingly, the introduction of three PO linkages at each wing of AON14 did not alter the AON efficacy or allele selectivity: AON16 reduces WT and A431 E PSEN1 protein levels to similar levels as AON14 (85% reduction of A431 E and 10% reduction ofWT) (Figure 3D). Similarly, the introduction of two PNms linkages within the positions +2 and +4 of the central part of the antisense oligonucleotide starting from 5’ to 3’in a full PS backbone (AON17 and AON18, SEQ ID NO:35 and 37) or in a mix PO/PS backbone (AON 19, SEQ ID NO:39) did not significantly impact the AON efficacy or allele selectivity: AON17, AON18 and AON19 reduce PSEN1 A431 E and WT protein levels at similar levels that AON14 (for PSENI A431 E: 79%, 82%, and 79%, respectively, while for WT PSEN1 : 1 %, 8%, and 24%, respectively) (Figure 3D).
Conclusions These results show that the best effectivity and specificity is obtained when the position of the nucleotide opposite the mutation is at +6, +7 or +8 of the oligonucleotide sequence starting from 5’ to 3’ (i.e, in the 3 first positions of the central part of the antisense oligonucleotide starting from 5’ to 3’), and that T>U substitution at that position increases allele selectivity while keeping a similar effectivity towards the mutant PSEN1 . In addition, the results show that the addition of PO and/or PNms linkages does not significantly impact the efficacy and allele selectivity.
Example 4
Introduction
Results in Example 3 show that AONs targeting human A431 E PSEN1 have the best effectivity and allele specificity when the position of the nucleotide opposite the mutation is at position +6, +7, or +8 of the oligonucleotide sequence starting from 5’ to 3’ (i.e, in the 3 first positions of the central part of the antisense oligonucleotide starting from 5’ to 3’), and that T>U substitution at that position increases allele selectivity while keeping a similar effectivity towards the mutant PSEN1 . The goal of this experiment is to determine if these criteria in AON design apply to another C/G/T > A mutation in PSEN1 . More than 150 different mutations in PSEN1 have been described in FAD patients. Of those, 4% are caused by C>A mutations (like A431 E), and 17% are caused by C/G/T > A mutations. A series of AONs targeting human S212Y (caused by C>A mutation) were designed and tested in fibroblasts transfected with plasmids containing either the Wt or S212Y mutant PSEN1 cDNA construct. AON efficacy and allele selectivity were asses on mRNA by Digital droplet (dd)PCR.
Material and Methods
AONs
AONs specifically targeting human S212Y PSEN1 mRNA were designed (see Table 5). AONs had a backbone containing phosphorothioate (PS) with a DNA gap flanked by 2’-MOE wings at 5’ and 3’ end. AONs were synthesized as explained in Example 3. Six AONs targeting A431 E were also tested in this experimental set-up (AON4, AON5, AON6, AON13, AON14 and AON15; see Table 4).
Table 5: Human AON sequences targeting S212Y
Figure imgf000163_0001
A=adenosine; G=guanine; T=thymine; C=cytosine; U=Uracil; C*=5-methylcytosine. D=DNA, e=2’-MOE, s=PS linkage. Column A= Position of mutation within the antisense oligonucleotide sequence starting from 5’ to 3’. Column B= Nucleotide opposing the mutation.
The corresponding sequences of the non-modified oligonucleotides are represented by SEQ ID NO: 173, 175, 177, 179, 181 , 183, 185, 187, 189, 191 , 193, 195, 197, 199.
Transfection of human PSEN1 expression vectors and AONs
NIH3T3 cells were transfected with plasmid encoding human wild-type (Wt) PSEN1 variant 1 (NM_000021 .4), mutant A431 E PSEN1 or mutant S212Y PSEN1 transcripts. See Example 3 for more details on cell culture and transfection procedures.
RNA extraction and cDNA synthesis
Twenty-four hours after transfection cells were harvested and total RNA was extracted with the RNAse Mini Kit (Ref:74104, Qiagen). DNAse treatment was done on the column during RNA extraction following manufacture’s instructions (Rnase-Free DNase Set, Ref:79254, Qiagen). 200ng of RNA were used as input for the cDNA synthesis using Superscript IV Enzyme (Thermo Fisher, Ref:18090050) and random hexamer primers (Sigma, 11034731001).
Digital droplet (dd)PCR analysis
Specific Taqman minor groove binder (MGB) assays were designed to detect human PSEN1 and NPT-II encoded in the plasmid (Table 6). Taqman assays were purchased from Applied Biosystems. Digital droplet PCR analysis was performed on 1 pl of cDNA (cDNA was diluted 20X for PSEN1 assay and 400X for NPT-II assay) in a 20pl reaction volume using an annealing/extension temperature of 60° C according to the manufacturer’s instructions (BioRad). mRNA copies of hPSENI were normalised to mRNA copies of NPT-II and expressed relative to non-AON treated samples. Data is expressed as a ratio of the relative levels of WT PSEN1 versus Mutant PSEN1.
Table 6. Taqman Assays hPSENI plasmid
Figure imgf000164_0001
Figure imgf000165_0001
Results
Initially, we tested if the effect in allele selectivity and efficacy described in Example 3 on protein by WES was also detectable on mRNA by (dd)PCR (Figure 4A). Data is shown as the ratio between the mRNA levels of WT and mutant PSEN1 . If the ratio is 1 , the AON has no allele selectivity. The higher the ratio is, the more allele preference towards the mutant has the AON. Figure 4A shows that AONs containing the mutation in positions +6, +7, or +8 of the oligonucleotide sequence starting from 5’ to 3’ (i.e, in the 3 first positions of the central part of the antisense oligonucleotide starting from 5’ to 3’) have 2 - 3 times more preference towards mutant A431 E than WT. In addition, the ratio WT I Mutant is higher in AONs that have a U opposing the mutation instead of T (Especially in positions +6 and +8 of the oligonucleotide sequence starting from 5’ to 3’).
Next, a set of AONs targeting S212Y PSEN1 with mutations in different positions and with a T>U substitution opposite to the mutation were designed and tested (Table 5). Results in Figure 4B show that AONs with the mutation in positions +6, +8 and +10 of the oligonucleotide sequence starting from 5’ to 3’ (i.e., in positions +1 , +3, +5 of the central part of the antisense oligonucleotide starting from 5’ to 3’), show the highest allele selectivity (Ratio Wt I Mutant is higher than 1 .5). In addition, T to U substitution opposite to the mutation improves allele selectivity in positions +6, +8, and +12 (i.e., in positions +1 , +3, +7 of the central part of the antisense oligonucleotide starting from 5’ to 3’).
Conclusions
These results show that the best effectivity and specificity are obtained when the position of the nucleotide opposite the mutation is between position +6 and +10 of the oligonucleotide sequence starting from 5’ to 3’ (i.e., in the first five positions of the central part of the antisense oligonucleotide starting from 5’ to 3’) in 2 different PSEN1 mutations. In addition, for both PSEN1 mutations, there are specific positions where a T > U substitution opposite to the mutation increases allele selectivity. Therefore, our newly defined criteria on AON design at least apply to two PSEN1 mutations from FAD patients (A431 E and S212Y). Example 5
Introduction
Neurons are one of the major cell types affected in FAD patients. In Example 3, a group of efficient and allele-selective AONs targeting A431 E PSEN1 was identified using mouse fibroblasts and plasmids containing either the Wt or A431 E mutant PSEN1 cDNA construct. To determine if selected AONs are also efficient and selective for mutant PSEN1 in a disease-relevant cell, neurons differentiated from FAD patient-derived iPSC were treated for 10 consecutive days with AON14 (SEQ ID NO:28), AON15 (SEQ ID NO:30), and AON16 (SEQ ID NO:33). All the selected AONs fulfil our newly established design criteria for best AON effectivity and allele selectivity: nucleotide opposite the mutation between positions +6 and +8 of the oligonucleotide sequence starting from 5’ to 3’ (i.e., first three positions of the central part of the antisense oligonucleotide starting from 5’ to 3’), and a T>U substitution on that position.
Material and Methods
AONs
See Table 4 in Example 3. iPSC differentiation to neurons and AON qymnotic uptake iPSC derived from a patient carrying A431 E PSEN1 mutation in heterozygosis were differentiated into neurons (cells generated by Bart de Strooper group). Briefly, iPSC were seeded as a monolayer in a Matrigel-coated 6-well plate and cultured for 12 days in Neuronal Induction Medium (NIM, see composition in Table 8). On Days in Vitro (DIV) 12, the monolayer of cells was mechanically dissociated and cells were seeded in a Matrigel-coated 6-well plate and Neuronal Maintenance Medium (N.M.M., see composition in Table 7) to induce neural progenitor differentiation. On DIV 24-26, Neural Progenitor Cells were dissociated and seeded for neuronal maturation in N.M.M medium on Poly-L-Ornithine-laminin-coated plates with N.M.M medium and treated for 48h with 10pM DAPT (Tocris; cat# 2634) on DIV31. On DIV36, Neural progenitor cells were split and seeded in a Poly-L-Ornithine-laminin-coated 96-wells plate with N.M.M medium for further neuronal maturation (30.000 cells/well). AON treatment started on DIV62, during 10 days 10 pM of AON14, AON15 or AON16 (Table 4) were added daily to the medium. On DIV71 neurons were harvest.
Table 7: Composition of Neural induction medium
Figure imgf000166_0001
Figure imgf000167_0001
Table 8: Composition of Neuronal induction medium (NIM)
Figure imgf000167_0002
RNA extraction and cDNA synthesis
Total RNA was extracted using RNeasy 96 QIAcube HT Kit (Ref: 74171 , Qiagen). DNAse treatment was done on the column during RNA extraction following the manufacture’s instructions (Rnase-Free DNase Set, Ref: 79254, Qiagen). A clean-up protocol was performed to detach the AON from the RNA. Briefly, RNA was mixed with RTL buffer and incubated for 5 min at 95°C followed by cool for 2 min on ice incubation. Then, RNA was purified using the RNeasy MinElute Cleanup Kit following the manufacture’s instructions (Ref: 74204, Qiagen). 200ng of RNA were used as input for the cDNA synthesis using Superscript IV Enzyme (Thermo Fisher, Ref: 18090050) and random hexamer primers (Sigma, 1 1034731001).
Digital droplet (dd)PCR analysis
SNP assays to detect human WT and A431 E PSEN1 were designed by BioRad (Assay ID hPSENI WT: dMDW4344923033; Assay ID hPSENI A431 E: dMDM4344923031). Taqman assays for housekeeping genes were purchased at Thermo Fisher: TFRC (Ref: Hs99999911_m1) and GAPDH (Ref: Hs04420632_g1). (dd)PCR analysis was performed on 2 pl of cDNA for PSEN1 and TFRC or 2 pl of 20X diluted cDNA for GAPDH in a 20pl reaction volume using an annealing/extension temperature of 55° C according to the manufacturer’s instructions (BioRad). mRNA copies of WT or A431 E PSEN1 were normalised to the copies of TFRC and GAPDH housekeeping genes. Data is expressed relative to the control sample with no AON treatment.
Results
Treatment of iPSC-derived neurons during 10 days with AON14, AON15 and AON16 (Table 4) results in a selective reduction of mutant A431 E PSEN1 mRNA levels. Mutant A431 E mRNA levels are reduced by 49%, 62% and 63%, respectively, while WT PSEN1 mRNA levels are reduced by 17%, 22% and 22%, respectively.
Conclusions Our results show that three AONs of the invention (AON14, AON15 and AON16), specifically reduce the levels of mutant A431 E PSEN1 in a disease-relevant cell line (neurons differentiated from FAD patient-derived iPSC).
Example 6
Introduction
Mutations in Psenl (like A431 E) are believed to reduce the functionality of y-secretase and increase the production of long Ap species (>Ap42), which leads to an abnormal Ap profile with increased Ap42/40 and Ap42/38 ratios (Szaruga M et al., Cell. 2017 Jul 27;170(3):443-456.e14, Petit D et al., Mol Psychiatry. 2022 Apr 1). To confirm that A431 E mutation increases the production of long Ap species (>Ap42) and induces an abnormal Ap profile in neurons, we repeated the experiments from Example 2 with an AON that targets specifically mouse WT Psenl (AON87, Table 9). Primary neurons derived from Psenl A431 E heterozygous mouse embryos (Psenl Wt/A431E; APPKi) were treated with AON87 for 7 consecutive days and the mRNA levels of WT and A431 Psenl , as well as, the levels of soluble Ap38, Ap40, and Ap42 peptides were quantified.
Material and Methods
AONs
For details about AON synthesis see Example 1 .
Table 9: Additional mouse AON sequences
Figure imgf000168_0001
A=adenosine; G=guanine; T=thymine; C=cytosine; C*=5-methylcytosine. d=DNA, e=2’-MOE, s=PS linkage. 0=PO linkage.
Corresponding non modified sequence of the oligonucleotide represented by SEQ ID NO: 201 , 203.
Primary mouse neuronal cultures, Gymnotic uptake, Protein extraction and Quantification of soluble AP38, AP40 and AP42: see Example 1 .
RNA extraction and cDNA synthesis: see Example 5
Real-time PCR (RT-qPCR):
Specific primers were designed to amplify mouse WT Psenl , A431 E Psenl , Actin and Rps23 (Table 10). RT-qPCR analysis was performed on 5ng of cDNA in a 15pl reaction volume using an annealing/extension temperature of 65.5° C and PowerUP SYBR green Master mix (Thermo Fisher, A25741). RT-qPCR was performed with ViiA 7 Real-Time PCR System (Thermo Fisher). Table 10: RT-qPCR primers
Figure imgf000169_0002
Results
Treatment of heterozygous neurons (Psen1A431/ wt;APPKi cells) with AON87 resulted in a dosedependent reduction of WT Psenl mRNA levels, which reached 67% reduction at the highest 10 |j.M AON concentration (Figure 6A). Interestingly, the mRNA levels of A431 E Psenl show a 20% increase. To investigate whether the observed reduction in WT Psenl and upregulation of A431 E Psenl leads to an impairment of the y-secretase activity and an abnormal I disease-like Ap profile the levels of soluble Ap38, Ap40 and Ap42 peptides were measured in the medium. Total Ap levels (Ap38 + Ap40 + Ap42) were not affected by AON87 treatment (Figure 6B). Importantly, AON87 treatment causes a 50% reduction of Ap38 levels and a 30% increase of Ap42 levels relative to non-treated samples (0
Figure imgf000169_0001
This shift in the Ap production results in a 2.5-fold increase in the ratio Ap42/Ap38.
Conclusions
These results confirmed that A431 E mutation in Psenl reduces the functionality of y-secretase and increases the production of long Ap species (>Ap42) in primary mouse neurons, which leads to an abnormal I disease-like increase of Ap42/38 ratio. Furthermore, the unexpected upregulation of A431 E mRNA levels after the knock-down of WT Psenl suggests that total levels of Psenl are tightly regulated in neurons, and that compensatory mechanisms might exist. Another interesting observation is that the magnitude of change in the levels of each Ap specie analysed is different, for example Ap38 shows the biggest effect compared to non-treated, while Ap40 is almost not altered. The potential compensatory mechanisms on mRNA levels and the different affectation of each Ap species strengthens the need to use holistic readouts to evaluate the therapeutic benefit of the AON treatment.
Example 7
Introduction
Example 1 shows that AON20 treatment reduces Psenl protein levels and the total production of Ap peptides in A431 E homozygous mouse primary neurons. To confirm that those changes are driven by a reduction of mutant Psenl mRNA levels, mouse primary neurons derived from Psenl A431 E homozygous embryos (Psen1A431/A431E; APPKi) were treated for 7 consecutive days with AON20 (Table 2) and AON88 (Table 9). After, mutant Psenl mRNA levels were quantified by RT- qPCR. (Figure 7A).
Similarly, the effectivity and allele selectivity of AON20 and AON88 was asses by performing the same experiment in mouse primary neurons derived from Psenl A431 E heterozygous embryos (Psen1wt/A431E; APPKi) (Figure 7B).
Material and Methods
See Example 1 and 6.
Results
AON20 and AON88 treatment to primary mouse homozygous neurons (Psenl A431/ A431E; APPKi cells) resulted in a dose-dependent reduction of A431 E PSEN1 mRNA levels, which reached 54% and 71 % reduction at the highest 10 JJ.M AON concentration, respectively (Figure 7A).
AON20 and AON88 treatment to primary mouse heterozygous neurons (Psenl Wt/A431E; APPKi cells) resulted in a dose-dependent reduction of A431 E PSEN1 mRNA levels, which reached, 64% and 71 % reduction at the highest 10 JJ.M AON concentration, respectively. Interestingly, the mRNA levels of WT PSEN1 showed a 12% and 19% increase at the highest AON concentration, respectively. (Figure 7B).
Conclusions
AON20 and AON88, AONs of the invention, are effective in reducing specifically mutant A431 E Psenl mRNA levels in mouse primary neurons. Therefore, the changes in Psenl protein levels and Ap peptide levels described in Example 1 are most likely driven by the AON effect on A431 E Psenl mRNA levels. Furthermore, the unexpected upregulation of WT mRNA levels observed after treatment of both AONs targeting mutant Psenl , supports the idea that Psenl levels are tightly regulated in neurons, and reinforce the need to use holistic readouts to evaluate the effect of the AON treatment.
Example 8
Introduction
Figure 7B shows that treatment with AON20 and AON88 reduces the mRNA levels of mutant Psenl in a dose-dependent manner in primary mouse heterozygous neurons (Psen1wt/A431E; APPKi cells). To evaluate if the observed changes in mRNA levels lead to a recovery of y- secretase functionality and therefore ameliorate Ap profile, the levels of soluble Ap38, Ap40, and Ap42 peptides were quantified in the supernatant of the same cultures as in Figure 7B (only the samples treated with 10pM of AON were analysed).
Material and Methods
See Example 1 and 6.
Results
AON20 and AON88 treatment to primary mouse heterozygous neurons (Psenl Wt/A431E; APPKi cells) does not alter total Ap levels (Figure 8A). Importantly, treatment with both AONs increases the levels of Ap38 compared to the untreated samples (16% and 35%, respectively). Ap42 levels show a tendency to decrease of 15% and 7%, respectively (Not statistically significant) (Figure 8B). These changes lead to a decrease in the Ap42/Ap38 ratio, which reaches a 25% and 31 % reduction relative to non-treated condition, respectively (Figure 8C).
Conclusions
AON20 and AON88, AONs of the invention, are effective in increasing short Ap peptide levels and improving the Ap42/Ap38 ratio in primary mouse neurons. Similarly to Example 6, the changes in Ap38 are more pronounced than in the other Ap peptides analysed. This observation strengthens the idea that holistic readouts are needed to evaluate the therapeutic benefit of the AON treatment.
Example 9
Introduction
The goal of this experiment is to test if one AON of the invention (AON20, Table 2) is efficient and allele selective in-vivo when injected into the mouse brain. To this end, Psenl wt/A43iE.^pp^j aqun mice received an intracerebroventricular (ICV) single injection of AON20. Two weeks later, animals were sacrificed and the hippocampal mRNA levels of Wt and A431 E Psenl were quantified by RT-qPCR.
Material and Methods
Mice treatment
Seven weeks-old Psen1wt/A431E ; APPKi mice received a single ICV injection of AON20 in the right ventricle. 150pg of AON20 were administered in 5pl at an injection velocity of 0.3pl/min. Mice in the vehicle group received 5pl of artificial CSF. Animals were sacrificed 2 weeks afterthe injection. The right hippocampus was isolated for further analysis.
RNA extraction and cDNA synthesis
Tissues were homogenized in 1 ml of RNA-Bee (Bio-Connect) by grinding in a MagNa Lyser using MagNA Lyser Green Beads (Roche). Total RNA was extracted from the homogenate based on the manufacturer’s instructions. RNA clean-up protocol, cDNA synthesis and RT-qPCR was performed as indicated in Example 6.
Results
Single ICV injection of AON20 results in a 44% reduction of A431 E Psenl mRNA levels in the hippocampus. Levels of WT Psenl mRNA are not statistically significantly changed.
Conclusions
AON20, AON of the invention, shows efficacy and allele selectivity in-vivo when injected into the mouse brain.

Claims

Claims
1. An antisense oligonucleotide which preferentially targets a mutated allele of a protein of the y-secretase complex when present in a cell comprising and preferably expressing said mutated allele.
2. An antisense oligonucleotide according to claim 1 , wherein the protein of the y-secretase complex whose allele is mutated is PSEN1 or PSEN2, preferably PSEN1.
3. An antisense oligonucleotide according to claim 1 or 2, wherein
- the oligonucleotide is able to preferentially silence, inactivate, break down, knock down, reduce or decrease mutated PSEN1 or PSEN2 transcripts (preferably mutated PSEN1 transcripts) and
- the oligonucleotide is not (or minimally) able to silence, inactivate, break down, knock down, reduce or decrease wild-type PSEN1 or PSEN2 transcripts (preferably wild-type PSEN1 transcripts).
4. An antisense oligonucleotide according to any one of claim 1 to 3, wherein the oligonucleotide is able to normalize, reverse or correct abnormal A042/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios, preferably the Ap42/38 and/or Ap43/40 ratios, more preferably the Ap42/38 ratio.
5. An antisense oligonucleotide according to any one of claims 1 to 4, wherein the protein of the Y-secretase complex is endogenously expressed by said cell.
6. An antisense oligonucleotide according to any one of the preceding claims, wherein the cell is a neuronal cell.
7. An antisense oligonucleotide according to any one of the preceding claims, wherein the mutation in human PSEN1 is selected from the group consisting of the most frequent PSEN1 mutations: P117L, M139T, M139V, M146I, H163R, G206A, P264L, E280A.L392V, A431 E, and S121Y, more preferably E280A and A431 E.
8. An antisense oligonucleotide according to any one of the preceding claims, wherein the mutation in human PSEN1 is selected from the group consisting of mutations caused by a substitution of a guanine, thymine, or cytosine by an adenosine in the coding sequence, resulting in one of the following mutations in the PSEN1 protein: R35Q, V94M, F105I, R108Q, L113Q, T1 16N, P117T, P117Q, E120K, E123K, M139K, M139I, V142I, M146I, V151 M, Y154N, L166H, L174M, 11 SON, G206S, G206D, G209R, G209E, S212Y, H214N, G217D, S230N, A231T, M233I, F237I, A246E, Y256N, V261 I, G266S, R269H, V272D, T274K, R278K, E280K, R358Q, A360T, S365Y, G378E, F386I, F386L, S390N, A396T, A409T, C410Y, G417S, L424H, A431 E, A434T, P436Q, preferably A431 E.
9. An antisense oligonucleotide according to any one of the preceding claims, wherein the antisense oligonucleotide is able to recruit RNaseH to silence, inactivate, knock down, break down, decrease or reduce the levels or expression of the targeted mutant allele.
10. An antisense oligonucleotide according to any one of the preceding claims, wherein the antisense oligonucleotide comprises 15 to 30 or 20 to 30 nucleotides including a central region of 5 to 15 consecutive DNA nucleotides and/or DNA nucleotides analogues flanked in each end by wing regions comprising 1 to 5 RNA nucleotides analogues.
11. An antisense oligonucleotide according to claim 10, wherein the mutation present in the mutated allele or transcript of PSEN1 or PSEN2 is targeted by the first, second, third, fourth, fifth, sixth, seventh, eighth, nineth, tenth, eleventh or twelfth nucleotide of the central region of the oligonucleotide.
12. An antisense oligonucleotide according to claim 10 or 11 , wherein:
1) the DNA nucleotides in its central part have not been modified and the RNA nucleotides in each of its wings have been modified comprising a modified internucleoside linkage preferably a phosphorothioate and/or phosphoramidate (more preferably mesylphosphoramidate) and/or a modified sugar (preferably a 2’-MOE, 2’-OMe and/or a locked nucleic acid (LNA) monomer) and/or a modified base (preferably a 5- methylcytosine), or
2) a DNA nucleotide in its central part has been modified and the backbone (i.e. at least one internucleoside linkage) comprises a phosphorothioate and/or phosphoramidate (preferably mesylphosphoramidate), and the RNA nucleotides in each of its wings have been modified comprising a modified internucleoside linkage preferably a phosphorothioate and/or phosphoramidate (more preferably mesylphosphoramidate) and/or a modified sugar (preferably a 2’-MOE, 2’-OMe and/or a locked nucleic acid (LNA) monomer) and/or a modified base (preferably a 5-methylcytosine).
13. An antisense oligonucleotide according to any one of the preceding claims, said oligonucleotide comprising SEQ ID NO: 2, 4, 6, 8, 7,8, 9, 10, 11 , 12, 14, 16, 18, 20, 22, 24, 25, 26, 27, 28, 29, 30, 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99, 101 , 103, 105, 107, 109, 1 11 , 113, 115, 117,122, 124, 126, 128,130,132,134,136,138,140,142,144, preferably SEQ ID NO: 7, 8, 9, 10, 11 , 12, 25, 26, 27, 28, 29, 30, 33, 34, 35, 36, 37, 38, 39, 47, 97, 99, 115 more preferably SEQ ID NO: 8, 10, 12, 26, 28, 30, 33, 35, 37, 39, 47, 97, 99, 115, 219, 221 , 223, 225, 227, 229, 231 , 233, 235, 237, 239, 241 , 243, 245, 247, 249, 251 , 253, 255, 257, 259, 261 , 263, 265, 267, 269, 271 , 273, 275, 277, and most preferably SEQ ID NO: 28, 30, 33, 37, 39, 47,85, 97, 99, 115, 221 , or 223.
14. A composition comprising the antisense oligonucleotide according to any one of claims 1 to 13 wherein the composition comprises a pharmaceutically acceptable excipient.
15. An antisense oligonucleotide according to any one of claims 1 to 13 or a composition according to claim 14 for use as a medicament.
16. An antisense oligonucleotide or a composition according to claim 15, for use as a medicament for treating and/or delaying and/or curing and/or preventing and/or ameliorating a disease or condition associated with an abnormal processing of the Amyloid Precursor Protein (APP), preferably familiar Alzheimer disease (FAD).
17. An antisense oligonucleotide or a composition for use according to claim 15 or 16, wherein the antisense oligonucleotide elicits at least one of the following:
- the oligonucleotide is able to preferentially silence, inactivate, break down, knock down, reduce or decrease mutated PSEN1 or PSEN2 transcripts (preferably mutant PSEN1 transcript) and the oligonucleotide is not (or minimally) able to silence, inactivate, break down, knock down, reduce or decrease wild-type PSEN1 or PSEN2 transcripts (preferably wild-type PSEN1 transcript),
- the oligonucleotide is able to normalize, reverse, or correct abnormal A042/38 and/or Ap43/40 and/or Ap43/37 and/or Ap42/40 ratios, preferably the Ap42/38 and/or Ap43/40 ratios, more preferably the Ap42/38 ratio, and
- the oligonucleotide is able to alleviate one or more symptom(s) and/or characteristic(s) and/or to improve a parameter linked with or associated with a disease or condition associated with an abnormal processing of the Amyloid Precursor Protein (APP), preferably familiar Alzheimer disease (FAD).
18. Method for treating and/or delaying a disease or condition associated with an abnormal processing of the Amyloid Precursor Protein (APP), preferably familiar Alzheimer disease (FAD) using an antisense oligonucleotide or a composition as defined in any one of claims 1 to 17.
19. An antisense oligonucleotide comprising 15 to 30 or 20 to 30 nucleotides including a central region of 5 to 15 consecutive DNA nucleotides and/or DNA nucleotides analogues flanked in each end by wing regions comprising 1 to 5 RNA nucleotides analogues, which preferentially targets a mutated allele of a protein of the y-secretase complex when present in a cell comprising and preferably expressing said mutated allele, wherein the mutation present in the mutated allele is targeted by the first, second, third, fourth, fifth, sixth, seventh, eight, nineth, tenth, eleventh, or twelfth nucleotide of the central region of the oligonucleotide.
20. An antisense oligonucleotide comprising 15 to 30 or 20 to 30 nucleotides including a central region of 5 to 15 consecutive DNA nucleotides and/or DNA nucleotides analogues flanked in each end by wing regions comprising 1 to 5 RNA nucleotides analogues, which preferentially targets a mutated allele of a protein of the y-secretase complex when present in a cell comprising and preferably expressing said mutated allele, wherein the mutation present in the mutated allele is targeted by the sixth, seventh, eighth, nineth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, or eighteenth nucleotide in the oligonucleotide sequence starting from 5’ to 3’.
21. An antisense oligonucleotide according to claim 10, wherein the mutation present in the mutated allele or transcript of PSEN1 or PSEN2 is targeted by the sixth, seventh, eighth, nineth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, or eighteenth nucleotide of the oligonucleotide sequence starting from 5’ to 3’.
22. An antisense oligonucleotide according to any one of claims 1-12, said oligonucleotide comprising SEQ ID NO: 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202 preferably SEQ ID NO: 178, 180, 182, 192, 194, 196, 198 more preferably SEQ ID NO: 192, 194, 196.
23. An antisense oligonucleotide according to claim 10, wherein the mutation present in the mutated allele or transcript of PSEN1 or PSEN2 is targeted by the first, second, third, fourth, or fifth nucleotide of the central region of the oligonucleotide.
24. An antisense oligonucleotide according to claim 10, wherein the mutation present in the mutated allele or transcript of PSEN1 or PSEN2 is targeted by the sixth, seventh, eighth, nineth, or tenth nucleotide of the oligonucleotide sequence starting from 5’ to 3’.
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